cytoconference.org isac-net.org
XXVIII Congress of the International Society for Advancement of Cytometry
Innovation, Discovery, and Translation May 19 – 22, 2013 San Diego Convention Center San Diego, California, USA
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ISAC is grateful for the contributions of the following sponsors for their generous support of CYTO 2013: GOLD LEVEL___________________________________________________
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ISAC XXVIII International Congress San Diego, California, USA May 19 – 22, 2013 Dear Colleagues, On behalf of the CYTO Organizing Committee and ISAC Council, it is our pleasure to welcome you to CYTO 2013, the XXVIII Congress of the International Society for Advancement of Cytometry. ISAC’s mission is to advance the science and practice of cytometry, the quantitative analysis of cells and cell systems. Cytometry encompasses multiple disciplines from the physical sciences and engineering to basic research and clinical practice, and CYTO is the signature venue for showcasing the state of the art and science of cytometry. San Diego, our host city, is a center of biomedical research, and provides an excellent back drop for the CYTO 2013 theme of Innovation, Discovery, and Translation. The CYTO Program includes elements for scientists working in all areas of cytometry and at all career stages. The CYTO 2013 Education Program starts on Saturday, May 18th, with Introductory Flow and Image Cytometry Courses, the Intermediate Course on Advanced Flow Cytometry Data Analysis, and a program of Scientific Tutorials providing focused updates on a range of research and clinical topics in flow and image cytometry, as well as core facility management. Also on Saturday, ISAC will host its 2nd CYTO-Innovation Forum, providing an opportunity to discuss the challenges and opportunities of commercializing new cell analysis technologies and applications. Industry experts, technology innovators, and others involved in the business of cytometry are welcome to participate in this pre-CYTO event. The CYTO 2013 Scientific Program opens Sunday, May 19th with the Wallace H. Coulter Centennial Lecture, “Spatial Systems Biology”, presented by Joe Gray, commemorating the 100th birthday of this pioneering inventor, entrepreneur, and philanthropist. The CYTO 2013 program continues with Frontiers and Plenary Sessions featuring cutting edge cytometry technology and applications, Parallel Sessions with examples of contemporary cytometry from the research lab to the clinic, and Poster Sessions that provide an opportunity for detailed discussions between authors and delegates. A diverse program of interactive workshops provides an opportunity for experts and novices alike to discuss and debate emerging or controversial issues in cytometry. The CYTO Commercial Exhibition features more than 70 companies displaying hardware, software, and reagents for cytometry research. The popular lunchtime Commercial Tutorials provide opportunities to hear in detail about new product offerings from leading companies. New this year is an Exhibitor Showcase, to be held Monday during lunch in the Exhibit Hall, giving vendors a forum to highlight new products. We appreciate the support of all of our exhibitors, and especially our Sponsors, for helping to make CYTO possible. We want to express our sincere thanks to the members of the CYTO Organizing Committee and to the 50+ members of the CYTO Program Committee, including the ISAC Scholars, who proposed themes and speakers, and assisted with abstract review. Thanks go also to our Course and Tutorial faculty, Workshop leaders, and Session Chairs for contributing their time and talents. Lastly, we want to thank the FASEB Office of Scientific Meetings & Conferences team and Michelle Butler, ISAC Executive Director, for their tireless work in ensuring that CYTO 2013 is a success. We encourage all members to participate in ISAC’s efforts to advance cytometry. Please attend the Business Meeting on Wednesday afternoon to hear about ISAC’s recent efforts and future plans, volunteer for service on committees and task forces, and share your thoughts on the future of the Society and cytometry with the ISAC Council and management at any time. Finally, enjoy CYTO and San Diego! John P. Nolan ISAC President-Elect ISAC 2013 Program and Abstracts
Larry A. Sklar CYTO 2013 Program Chair 1
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Table of Contents Sponsors and Supporters.................................................................................................................... Inside Front Cover Welcome Letter............................................................................................................................................................1 ISAC 2012 – 2014 Executive Committee and Councilors................................................................................................... 5 ISAC Leadership and Congress Organizers...................................................................................................................6 Isac Special Committees & Task Forces........................................................................................................................8 General Information Registration .........................................................................................................................................................11 Badge/Access Control..........................................................................................................................................11 Business Services.................................................................................................................................................11 Cell Phones.........................................................................................................................................................11 Child Care...........................................................................................................................................................11 Closing Reception at the Stingaree.......................................................................................................................11 Commercial Exhibits – Exhibit Hall Hours...........................................................................................................12 Commercial Tutorials...........................................................................................................................................12 Companion/Guest Registration............................................................................................................................12 Concierge – San Diego Convention Center..........................................................................................................12 Cyber Café ..........................................................................................................................................................12 CMLE...................................................................................................................................................................12 CYTO Innovation.................................................................................................................................................12 Dining Options....................................................................................................................................................13 Disabilities and Special Needs.............................................................................................................................13 Exhibitor Showcase..............................................................................................................................................13 First Aid and Emergencies....................................................................................................................................13 ICCE....................................................................................................................................................................13 Internet/Wireless Access......................................................................................................................................13 ISAC Booth..........................................................................................................................................................13 Message Board....................................................................................................................................................13 Mobile APP.........................................................................................................................................................13 Poster and Multimedia Presentations....................................................................................................................13 Pre-Congress Courses..........................................................................................................................................14 Practical Information for San Diego.....................................................................................................................14 Recording............................................................................................................................................................15 Scientific Tutorials................................................................................................................................................15 Speaker Ready Room...........................................................................................................................................15 Transportation......................................................................................................................................................15 City Map....................................................................................................................................................................16 San Diego Convention Center Floor Plans..................................................................................................................17 Service Location & Telephone Numbers.....................................................................................................................19 Committee Meetings...................................................................................................................................................20 Congress Overview....................................................................................................................................................21 Special Lectures..........................................................................................................................................................24 Daily Program Saturday, 18 May.................................................................................................................................................25 Sunday, 19 May...................................................................................................................................................27 Monday, 20 May..................................................................................................................................................31 Tuesday, 21 May..................................................................................................................................................36 Wednesday, 22 May............................................................................................................................................41 Multimedia and Poster Sessions..................................................................................................................................45 Commercial Tutorials & Exhibits Commercial Tutorials...........................................................................................................................................66 Exhibitor Showcase..............................................................................................................................................75 Exhibitor Listing...................................................................................................................................................76 Exhibit Hall Floor Plan.........................................................................................................................................77 Exhibiting Companies..........................................................................................................................................78 Oral Session Abstracts................................................................................................................................................90 Poster Session Abstracts............................................................................................................................................135 Speaker/Author Index...............................................................................................................................................235 Program-at-a-Glance..........................................................................................................................Inside Back Cover
ISAC 2013 Program and Abstracts
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Download the CYTO 2013 mobile app via iPhone/iPad and Android native apps or via Blackberry, Windows Phone, and your desktop through the mobile web.
To access the CYTO 2013 mobile app online, visit http://cyto2013.crowdcompass.com/
ISAC 2012 – 2014 Executive Committee and Councilors
John P. Nolan President La Jolla Bioengineering Institute
Andreas Radbruch President-Elect Deutsches RheumaForschungszentrum Berlin
Paul J. Smith Past President Cardiff University
Timothy Bushnell, Secretary University of Rochester
Paul K. Wallace, Treasurer RPCI Laboratory of Flow Cytometry
Derek C. Davies London Research Institute, Cancer Research UK
Grace Marie Chojnowski Queensland Institute of Medical Research
Andrea Cossarizza University of Modena and Reggio Emilia
Mario Roederer Vaccine Research Center, NIAID, NIH
Ryan Brinkman British Columbia Cancer Agency
Peter A. Lopez New York University
ISAC 2013 Program and Abstracts
Rachel J. Errington Cardiff University
Rui Gardner Instituto Gulbenkian de Ciência
Joanne Lannigan University of Virginia
Alfonso Blanco-Fernandez University College of Dublin
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ISAC Leadership and Congress Organizers ISAC 2012 – 2014 Executive Committee John P. Nolan, President La Jolla Bioengineering Institute Andreas Radbruch, President-Elect Deutsches Rheuma-Forschungszentrum Paul J. Smith, Past President Cardiff University Timothy Bushnell, Secretary University of Rochester Paul K. Wallace, Treasurer Roswell Park Cancer Institute
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Bruce Edwards University of New Mexico Virginia Litwin Covance Central Laboratory Services Zofia Maciorowski Curie Institute Robert Murphy Carnegie Mellon University John Nolan La Jolla Bioengineering Institute
ISAC 2012 – 2014 Councilors
Jeff Price Vala Sciences Inc. and Sanford Burnham Medical Research Institute
Alfonso Blanco-Fernandez University College of Dublin
Andreas Radbruch Deutsches Rheuma - Forschungszentrum
Ryan Brinkman British Columbia Cancer Agency
Mario Roederer Vaccine Research Center, NIAID, NIH
Grace Marie Chojnowski Queensland Institute of Medical Research
Paul Smith Cardiff University
Andrea Cossarizza University of Modena and RE
Paul Wallace Roswell Park Cancer Institute
Derek C. Davies London Research Institute, Cancer Research UK
CYTO 2013 Program Committee
Rachel J. Errington Cardiff University
Mehrnoosh Abshari National Institutes of Health
Rui Gardner Instituto Gulbenkian de Ciência
Nima Aghaeepour British Columbia Cancer Agency
Joanne Lannigan University of Virginia
Donat Alpar University of Pecs
Peter A. Lopez New York University
Alireza Ardjmand The University of Newcastle
Mario Roederer Vaccine Research Center, NIAID, NIH
Kewal Asosingh Cleveland Clinic
CYTO 2013 Organizing Committee
David Basiji Amnis Corp
Larry Sklar, Chair University of New Mexico
Anne Carpenter Broad Institute of Harvard & MIT
Ryan Brinkman British Columbia Cancer Agency
Pratip Chattopadhyay Vaccine Research Center, NIAID, NIH
Tim Bushnell University of Rochester
Estelle Glory-Afshar Carnegie Mellon University
ISAC 2013 Program and Abstracts
Sung Hwan Cho Nano Collect, Inc.
Ricardo Morilla The Institute of Cancer Researach
Scott Cram Los Alamos National Laboratory
Shazib Pervaize National University of Singapore
Derek Davies Cancer Research UK
Katarzyna Piwocka Nencki Institute of Experiemental Biology
Albert Donnenberg University of Pittsburgh Medical Center
Kylie Price Malaghan Institute
Vera Donnenberg University of Pittsburgh Medical Center
Bartek Rajwa Purdue University
David Galbraith University of Arizona
Paul Robinson Purdue University
Steven Graves University of New Mexico
Gustavo Rohde Carnegie Mellon University
Philip Hexley Shriners Hospitals for Children, Cincinnati
Gergely Toldi Semmelweis University
Dayong Jin Macquarie University
Rachael Walker University of Cambridge
Tomas Kalina Charles University in Prague
Robert Zucker U.S. Environmental Protection Agency
Joanne Lannigan University of Virginia
ISAC Executive Office
Anis Larbi Singapore Immunology Network Thomas Laurell Lund University James F. Leary Purdue University Silas Leavesley University of South Alabama Michael Lewis University of Vermont Er Liu La Jolla Bioengineering Institute Gerard Lizard University of Bourgogne Stephen Lockett NCI - Frederick/SAIC
Michelle Butler, Executive Director 9650 Rockville Pike Bethesda, MD 20814 USA Tel. 301-634-7454,
[email protected]
CYTO Meeting Management Marcella Jackson, Director Roya Jaseb, Meeting Manager Taylor Shaw, Meeting Assistant Janet Kearney, Exhibit Manager Joni Friedman, Exhibit Coordinator Josie Leftwich, Registrar FASEB Office of Scientific Meetings & Conferences 9650 Rockville Pike Bethesda, MD 20814 USA Tel. 301-634-7010;
[email protected]
Peter Lopez NYU School of Medicine Barry Moran Trinity College Dublin
ISAC 2013 Program and Abstracts
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Isac Special Committees & Task Forces Task Forces & Committees 2012 - 2014 Members Certification Advisory Committee Robert Murphy, Chair Mike Borowitz, Vice Chair Derek Davies Bruce Greig Joanne Lannigan Mara Neal Paul Robinson Ulrich Sack Elizabeth Stone Brent Wood John Nolan (ex officio) Andreas Radbruch (ex officio)
Core Managers Task Force Derek Davies, Chair Julie Auger Kathy Brundage Paula Campbell Grace Chojnowski Benjamin Daniel Ian Dimmick Elmar Endl Marie Follow Anna Fossum Rui Gardner Enid Keyser Desiree Kunkel Kathy Heel Richard Konz Zip Kruger-Gray Charles A. Kuszynski Joanne Lannigan Peter Lopez Lola Martinez Simon Monard Carol Oxford Greg Perry Joel Puchalski Andy Riddell Alan Saluk Kathleen Schell Adrian Smith Greg Veltri
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Rachael Walker Maggie Wang Kevin Weller John Nolan (ex officio) Andreas Radbruch (ex officio)
Council of ISAC Associated Societies Grace Chojnowski, Chair Gerald Lizard, Vice Chair Mike Keeney Oscar Segurado Janos Szollosi Ivan Vorobjev
Education Zosia Maciorowski, Chair Pratip Chattopadhyay Awtar Krishan Jonni S. Moore Gustavo Rohde John Nolan (ex officio) Andreas Radbruch (ex officio)
Elearning Delivery Task Force Pratip Chattopadhyay, Leader Nima Aghaeepour Kewal Asosingh Alfonso Blanco Ryan Brinkman Lara Kreps Mike Ormerod Alan Saluk
Flow Cytometry Content Task Force Jonni Moore, Leader Grace Chojnowski Derek Davies Peter Lopez Phil McCoy Mario Roederer Joe Trotter Paul Wallace Jennifer Wilshire
ISAC 2013 Program and Abstracts
Flow Cytometry Data Standards Task Force Ryan Brinkman, Chair Kim RM Blenman Chris Bray James Cavenaugh Francisco Chang César Collino Nicholas Crosbie Chakradhar Dunna Michael Goldberg Mark Hubbard Bill Hyun Simon Lange Ray Lefebvre Robert Leif Wayne Moore David Novo Leo Ostruszka David Parks Barclay Purcell Josef Spidlen Adam Treister Jim Wood Rober Zigon Bob Zucker John Nolan (ex officio) Andreas Radbruch (ex officio)
FlowRepository Steering Committee John Nolan, Interim Chair Ryan Brinkman Jeannine Holden Wayne Moore Mario Roederer
Finance Paul Wallace, Chair Rachel Errington Tomas Kalina Silas Leavesley Peter Lopez Bartek Rajwa John Nolan (ex officio) Andreas Radbruch (ex officio)
ISAC 2013 Program and Abstracts
Image Cytometry Content Task Force Gustavo Rohde, Leader Rachel Errington Stephen Lockett Anil Parwani Bartek Rawja Gyorgy Vereb
ISAC Scholars Review Committee Alex Nakeff, Chair Ryan Brinkman Tim Bushnell Zbigniew Darzynkiewicz Rachel Errington Stephen Lockett Zosia Maciorowski Paul Robinson Gustavo Rohde Mario Roederer John Nolan (ex officio) Andreas Radbruch (ex officio)
Live Education Delivery Task Force Awtar Krishan, Leader Umebreen Ahmad Gulderen Yanikkaya Demirel Paresh Jain H. Krishnamurthy Kovit Pattanapanyasat Alan Saluk Vivek Tanavde Bill Telford Qianjun Zhang
Membership Services Tim Bushnell, Chair Sung Hwan Cho Grace Chojnowski Peter Lopez Bruno Paredes Rachael Walker Nicole White John Nolan (ex officio) Andreas Radbruch (ex officio)
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Robert Hooke Distinguished Lecture & Awards Committee Paul Smith, Chair Robert Murphy Paul Robinson Ian T. Young Alan Waggoner John Nolan (ex officio) Andreas Radbruch (ex officio)
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Scientific Communications Andrea Cossarizza, Chair Nima Aghaeepour Zbigniew Darzynkiewicz Elmar Endl Enrico Lugli Jose Enrique O’Connor Mario Roederer John Nolan (ex officio) Andreas Radbruch (ex officio)
ISAC 2013 Program and Abstracts
General Information All Congress activities will be held at the San Diego Convention Center located at 111 W. Harbor Drive, San Diego, CA 92101, unless noted otherwise. CYTO Registration, Exhibits/ Posters, and the First Aid office are located on the Ground Level of the Convention Center. Sessions, Committee Meetings, Speaker Ready Room and the Meeting Management Office are located on the Upper Level of the Convention Center. Participation in CYTO 2013 is limited to registered delegates. Full Congress registration includes admission to all sessions, exhibits, coffee breaks, happy hours, Opening Reception, and exchange coupon for the Closing Reception at the Stingaree. There is an additional fee for the Pre-Congress Courses, CYTO Innovation, and the Scientific Tutorials on Saturday, 18 May at the San Diego Convention Center.
Congress Registration – Lobby
Registration for CYTO 2013 will be open during the following days and hours: Saturday, 18 May............................. 1100 – 1800 Sunday, 19 May.................................. 700 – 1900 Monday, 20 May................................ 730 – 1830 Tuesday, 21 May................................. 730 – 1830 Wednesday, 22 May........................... 730 – 1700 Refund Policy: Refunds will not be issued after 15 April, 2013
Exhibitor Registration – Lobby
Exhibitor registration is for booth personnel and provides admittance into the Exhibit Hall only. Exhibitor registration will be open during the following days and hours: Saturday, 18 May............................. 1100 – 1800 Sunday, 19 May.................................. 700 – 1900 Monday, 20 May................................ 730 – 1830 Tuesday, 21 May............................... 1030 – 1830 Wednesday, 22 May......................... 1030 – 1700
Badges/Access Control
Participation in CYTO 2013 is limited to registered attendees. The official badge is required for admittance to all sessions, social
ISAC 2013 Program and Abstracts
activities and the Exhibit Hall. A fee may be charged to reissue lost or misplaced badges. Please do not place a business card into the badge holder as identification. If there is an error on a badge, please have it corrected at the registration desk.
Business Services – Lobby
A FedEx Office as well as on-site full-service business center providing fax, copying, express mail, packaging, and printingis is located on the ground level of the Convention Center.
Cell Phones
Cell phone use is prohibited. Please turn off all cell phones and pagers prior to entering a session room. If you must leave a session early, please use the rear entrance and exit quietly.
Child Care
Please check with your hotel’s front desk or concierge service for names of babysitters who can provide care in your hotel room. Parents and guardians are required to perform their own reference checks and arrange child care independently. ISAC is not responsible for child care or the quality of care provided.
Closing Reception at the Stingaree
The Closing Reception will take place at the Stingaree, a night club located in the heart of the Gaslamp District (only a few blocks from the Convention Center). Enjoy a night of fun, music, dancing, and refreshments with friends and colleagues. the Stingaree also has a rooftop lounge providing a relaxing atmosphere. The event will take place on Wednesday, 22 May, 2013, from 1900 until 2300. Full registration includes an exchange coupon for the Closing Reception at the Stingaree. The Closing Reception coupon must be exchanged for an actual ticket on-site at the registration desk beginning Saturday, 18 May, 2013 until Monday, 20 May, 2013. Coupons will be exchanged on a first come first served basis until maximum capacity is reached. A ticket is required for admittance. Be sure to exchange your coupon early so you don’t miss out on the fun! 11
Commercial Exhibits – Exhibit Hall GH
Visit the commercial exhibits featuring displays by leading suppliers and vendors. A complete directory of exhibiting companies as well as the Exhibit Hall floor plan is located under the Exhibits tab of this program. Exhibits will be open during the following days and hours: Monday, 20 May.............................. 1130 – 1900 Tuesday, 21 May............................... 1130 – 1900 Wednesday, 22 May......................... 1130 – 1630 Note: Children under the age of 16 are not permitted in the Exhibit Hall without parent or guardian supervision.
Commercial Tutorials
21 commercial tutorial sessions are offered from 1245 – 1345 on Sunday, 19 May, and 1215 – 1315 on Tuesday, 21 May, and Wednesday, 22 May. Please refer to the Commercial Tutorial tab of this program for a complete list of offerings.
Companion/Guest Registration
Registered attendees of the CYTO 2013 Congress may sign up a spouse/guest as a Companion for $150 USD. Companion registration allows entrance to the Opening Reception, Happy Hour and the Closing Reception only. The Opening Reception is scheduled to be held Sunday, 20 May, 2013, Happy Hours are on Monday, 20 May, 2013 and Tuesday, 21 May, 2013, and the Closing Reception is on Wednesday, 22 May, 2013. Companion registrants are not permitted in the session rooms or the Exhibit Hall at any other time.
Concierge – Lobby
The Restaurant and Concierge Desk is located in Lobby E of the Convention Center. It will be open Saturday, 19 May, 2013, through Wednesday, 22 May, 2013, from 900 – 1800. A variety of services will be offered, including restaurant reservations, city information, map, brochures, directions, and the purchase of attraction tickets. Visit the Congress website to print coupons, or present your Congress badge at select locations to receive discounts. For baseball fans interested in seeing a Padres game, Petco Park is a short walk from the 12
Convention Center. For more information visit sandiego.padres.mlb.com.
Cyber Café – Exhibit Hall GH
For your convenience, ISAC has set up several computers with free Internet access in the Cyber Café. Attendees may use computers to browse the Internet and/or to check email. In consideration of others, please limit your use to 15 minutes. The Cyber Café will be open during the Commercial Exhibit hours for attendee use beginning Monday, 20 May, 2013.
CMLE
This Continuing Medical Laboratory Education activity is recognized by the American Society for Clinical Pathology as meeting the criteria for 23.5 hours of CMLE credit. ASCP CMLE credit hours are acceptable to meet the continuing education requirement for the ASCP Board of Registry Certification Maintenance Program. If you’re interested in earning CMLE credits, please follow these steps: 1. Be sure to add your name to the sign in sheet which will be located at the back of each session room (sign in sheets will be present in every session that is eligible to earn CMLE credits); 2. Complete an evaluation form for each session you attend and leave it in the box at the back of the room, immediately following that session; 3. Use the CMLE form available at registration to track the sessions you attend. Please follow the instructions on the form to finalize the process. You will need to drop off your completed form at the Meeting Management Office (Room 28B) before leaving the Congress. CMLE certificates will be issues by the ISAC Executive Office upon request via email to
[email protected].
CYTO Innovation
ISAC presents CYTO Innovation 2013, a forum for discussing the challenges and opportunities for the development and commercialization of cell analysis technologies (see page 25 for full description). The registration fee for this session is $75 USD.
ISAC 2013 Program and Abstracts
Dining Options
Internet/Wireless Access
Disabilities and Special Needs
Attendees may purchase “Instant Internet” for 24 hours at a rate of $12.95 per device. Purchasing the “Instant Internet” will provide you with access everywhere inside the Convention Center, including session rooms, with the exception of the Exhibit Hall.
Starbucks and Mrs. Fields will be open throughout the week at the convention center. A light lunch will be provided to congress registrants in Exhibit Hall GH on Monday, 20 May at 1200. The concession stand in Exhibit Hall GH will be open for the purchase of lunch or snacks on Tuesday, 21 May and Wednesday 22, May from 1100 – 1400. If you have a disability or special need that may have an impact on your participation in the meeting, please contact the Meeting Management Office. ISAC cannot ensure the availability of appropriate accommodations without prior notification of need.
Exhibitor Showcase – Exhibit Hall GH The exhibitor Showcase will take place on Monday, 20 May, 2013, from 1200 – 1330. The showcase will include presentations by several exhibiting companies.
Complimentary WiFi Service is available in the ground level lobby areas of the Convention Center.
ISAC Booth – Exhibit Hall GH
Please visit the ISAC booth to learn more about society activities and membership. The ISAC Executive Director will be available to answer your questions, as will the editor-in-chief of Cytomerty Part A during the Exhibitor Showcase on Monday, 20 May.
Message/Announcement Board
Messages can be posted on the board located in the registration area. Participants should check each day for messages.
First Aid and Emergencies – Lobby
Mobile APP
Saturday, 18 May............................... 830 – 1800 Sunday, 19 May.................................. 830 – 1930 Monday, 20 May................................ 830 – 1930 Tuesday, 21 May................................. 830 – 1930 Wednesday, 22 May........................... 830 – 1800
Poster and Multimedia Sessions – Exhibit Hall GH
The First Aid Office is located in Show Office G on the ground level near the registration area and will be open during the following days and hours:
ICCE
ISAC is an approved provider of continuing education for the ICCE certification. Any one hour of ISAC educational programming is worth one credit. Certified cytometrists will be asked to report their credit hours earned when they renew their certification. CYTO 2013 is worth a total of 23.5 credit hours. For more information on the International Cytometry Certification Examination and how to become a certified cytometrist, visit http://cytometrycertification.org.
ISAC 2013 Program and Abstracts
NEW!
Download the CYTO 2013 mobile app via iPhone/iPad and Android native apps or via Blackberry, Windows Phone, and your desktop through the mobile web. To access the CYTO 2013 mobile app online, visit http://cyto2013.crowdcompass.com/
Over 250 poster presentations will be on display in the Exhibit Hall. Please refer to the Poster Board Map in the Congress Addendum for the assigned location of presentations. Please refer to the schedule below for viewing hours. Monday, 20 May 700 – 1100 Authors must set up posters on assigned board 1130 – 1900 Poster Viewing 1715 – 1845 Poster Session 1 (Authors of odd numbered boards must be at their poster to answer questions and discuss their presentation.)
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Tuesday, 21 May 700 – 1900 Poster Viewing 1715 – 1845 Poster Session 2 (Authors of even numbered boards must be at their poster to answer questions and discuss their presentation.) Wednesday, 22 May 700 – 1600 Poster Viewing 1500 – 1600 Poster Session 3 (All authors present) Posters must be removed by 1630 on Wednesday, 27 May Outstanding Poster Awards All poster presenters who are students or postdoctoral researchers (who have received their doctorate within the last five years) are eligible. The posters will be judged at the time they are scheduled to be presented by the author. Those posters not attended at their scheduled time will not be considered. The names of authors selected for this award will be posted on the Message/Announcement Board in the Registration Area on Wednesday, 22 May. Poster winners will be presented a prize and recognized at the Awards Ceremony from 1645 – 1730 on Wednesday, 22 May.
Pre-Congress Courses
The Introduction to Image-Based Cytometry, Introduction to Flow-Based Cytometry, and the Advanced Data Analysis Course will be held on Saturday, May 18. For full description of the courses, please visit http://cytoconference.org/ cyto/2013/Pre-Congress-Courses.aspx. There is a separate registration fee of $150 USD for each of the Intro Courses and a fee of $300 USD for the Advanced Course.
Practical Information for San Diego Language The official language of the Congress is English. Translation services will not be provided.
Banking and Foreign Exchange The official currency in the United States is US Dollars. The US Dollar ($) is divided into 100 cents; notes are in denominations of $100, 50, 20, 10, 5, and 1. Banking hours in the United States are generally Monday through Friday, 900 – 1700. A few banks are open on Saturdays
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as well. You can withdraw money from ATM bank machines using your credit card and make purchases in stores and restaurants. Signs display accepted credit cards or you can ask the retailer. Check with your local financial institution to verify if you are able to use your home bank card or credit card prior to travel. ATM bank machine service is available in the lobby of The San Diego Convention Center. There are several banking institutions within walking distance of The Convention Center as well. Travelex offers a currency exchange kiosk in Terminal 1 at the San Diego International Airport. They also offer a mobile currency exchange card in Terminal 2 East. Services include travel insurance, traveler’s checks, money transfer, notary, facsimile, copy, and phone cards. International Currency Converters: www.GoCurrency.com www.Oanda.com Climate San Diego enjoys beautiful weather year round. San Diego’s average high temperature is 70°F and average low temperature is 59°F with 300 days of sunshine year round. Time Zone During CYTO 2013 San Diego will be on Pacific Savings Time. Electricity Voltage in the United States is 120 voltage (60 HZ). Outlet sockets use either a Type A plug which is a class II ungrounded plug with two flat parallel prongs, or a Type B plug which is a class I plug with two flat parallel prongs and a grounding pin. Dialing Codes The International access code for the United States is 1. The area code in San Diego is 619. While in the United States, you can call the directory information service number 411 for assistance in finding a phone number. Calls to area codes 800, 877, and 866 are toll-free in the United States. To make an international call, dial 011 followed by the country code, city code, and telephone number.
ISAC 2013 Program and Abstracts
Recording
Recording any presentation or session (oral or poster) by any means (photographing, audio taping, videotaping) is prohibited, except by an ISAC authorized agent for official purposes or by first authors who want to photograph their own poster presentations. All other photography and/ or videotaping is prohibited in the Exhibit Hall.
Scientific Tutorials
Saturday,18 May, is dedicated to the scientific tutorial program. Fifteen ninety-minute tutorials will be offered. A registration fee of $80 USD per tutorial (or $145 USD for two tutorials; $200 USD for three tutorials) is required to participate in the program. You may purchase tutorial tickets at the registration desk.
Speaker Ready Room – Room 28A
All speakers are requested to go to the Speaker Ready Room to review and check the compatibility of their presentation at least 4 hours prior to their session. Speakers must arrive in the session room 30 minutes prior to the scheduled start of their session to allow the operator time to load their presentation onto the computer. The operator will be seated at the table next to the stage. ISAC is not responsible for slides, laptops, or cables left in session rooms. Speakers are not required to bring a laptop. All session rooms will be equipped with a data projector and PC. Please bring your presentation on a Windows reusable USB flash drive or CDROM. We recommend that you bring a backup presentation format.
Shuttles & Taxi Service Shuttle services are a convenient way to navigate the city and are ideal for airport pickup or traveling to and from the Convention Center. Some shuttle services offer site-seeing tours of San Diego. Taxicabs are located at the airport, most hotels, attractions, and shopping centers. Base and fare rates will be displayed on the meter and will include a flag drop charge plus a per-mile and/or a per-hour charge. Approximate Taxi Cab Rate 2013: ••Getting in: $2.80 ••Each mile: $3.00 ••Per hour waiting time: $24.00 ••From airport: $1.50 Coronado Ferry The Ferry runs regularly across San Diego Bay, providing a gorgeous view of the downtown skyline and Coronado Bridge. A Ferry Stop is located right behind the Convention Center at the San Diego Harbor Excursion Kiosk. Water Taxi San Diego Water Taxi offers on-call transportation services in San Diego Bay. Travel serenely from your hotel to local shopping centers and restaurants. For more information about transportation or tour options, please visit the concierge desk in the lobby at the San Diego Convention Center or your hotel’s concierge service.
Transportation
San Diego Trolley If you’re staying in the downtown San Diego area, the iconic bright red trolley will take you around the city. The San Diego Trolley provides transportation to key downtown locations including The Convention Center. Trolley fares and passes are available at sdmts.com. Pedicabs & Carriages These are two popular forms of transportation along downtown’s waterfront and in the Gaslamp Quarter. There are also tours available, a unique way to explore the city.
ISAC 2013 Program and Abstracts
15
City Map
CYTO 2013 May 19, 2013 to May 22, 2013
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7th Ave.
6th Ave.
5th Ave.
4th Ave.
3rd Ave.
2nd
C St.
Broadway
Broadway
E St.
Pacific Hwy.
Harbor Drive
12th Ave.
Civic Center
11th Ave.
B St.
3 Ferry/ Tour Boats
1st
A St.
10th Ave.
4
Front St.
Ash
Union St.
San Diego Bay
State St.
Kettner Blvd.
Beech
Columbia St.
Little Italy
Cedar
India St.
Embarcadero
Date St.
F St.
E St.
2
Horton Plaza
F St.
G St.
G St.
Gaslamp Quarter
Market Street
Island Ave.
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VISITSANDIEGO.COM 619.525.5000
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3 W San Diego 1 Omni San Diego Hotel – 675 L St., San Diego, CA 92101 4 Wyndham San Diego Bayside
0.58 (in miles) from SDCC Distance 0.80
(619-231-6664)
52 Stingaree $:50$MPTJOH3FDFQUJPO
Ramada Inn - Gaslamp – 830 6th Ave., San Diego, CA 92101
1.10 0.30
(619-231-8307)
(619-398-3100)
5 Stingaree – the venue of CYTO’s Closing Reception – 454 6th Ave., San Diego, CA 92101
0.12 0.58 0.80
4 Wyndham San Diego Bayside (formerly Holiday Inn) – 1355 North Harbor Dr., San Diego, CA 92101
16
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3 W San Diego – 421 W B St., San Diego, CA 92101
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(619-544-9500)
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ISAC 2013 Program and Abstracts
ISAC 2013 Program and Abstracts
17
ESCALATOR TO CYTO SESSIONS UPPER LEVEL
ELEVATOR TO UPPER LEVEL
WIFI HOT SPOT
CYTO Registration
LOBBY
CYTO Exhibits & Posters
Concessions
FIRST AID
VISIT SAN DIEGO CONCIERGE
BOX OFFICE E
DESK
San Diego Bay
Ground Level Exhibition Halls GH
San Diego Convention Center
GROUND LEVEL EXHIBIT HALLS GH
ESCALATOR TO CYTO SESSIONS UPPER LEVEL
ESCALATORS TO CYTO EXHIBITS/POSTER GROUND LEVEL
SPEAKER READY ROOM
MEETING MANAGEMENT OFFICE
ELEVATOR TO GROUND LEVEL
San Diego Bay
Upper Level Session Halls
San Diego Convention Center
UPPER LEVEL SESSION ROOMS
18
ISAC 2013 Program and Abstracts
ESCALATORS TO CYTO EXHIBITS/POSTER GROUND LEVEL
Service Location & Telephone Numbers Meeting Management Office – Room 28B, Upper Level – (Tel. 619-525-6241) Saturday, 18 May 1200 – 1700 Sunday, 19 May 730 – 1700 Monday, 20 May 730 – 1700 Tuesday, 21 May 730 – 1700 Wednesday, 22 May 730 – 1700 Congress Registration & Information – Ground Level, Lobby – (Tel. 619-525-6244) Saturday, 18 May 1100 – 1800 Sunday, 19 May 700 – 1900 Monday, 20 May 730 – 1830 Tuesday, 21 May 730 – 1830 Wednesday, 22 May 730 – 1700 Exhibitor Registration & Information – Ground Level, Lobby – (Tel. 619-525-6245) Saturday, 18 May 1100 – 1800 Sunday, 19 May 700 – 1900 Monday, 20 May 730 – 1830 Tuesday, 21 May 1030 – 1830 Wednesday, 22 May 1030 – 1700 Speaker Ready Room – Room 28A, Upper Level – (Tel. 619-525-6242) Saturday, 18 May 1200 – 1700 Sunday, 19 May Monday, 20 May Tuesday, 21 May Wednesday, 22 May
730 – 1700 730 – 1700 730 – 1700 730 – 1700
First Aid Room – Show Office G, Ground Level, Lobby – (Tel. 619-525-6243) Saturday, 18 May 830 – 1800 Sunday, 19 May 830 – 1930 Monday, 20 May 830 – 1900 Tuesday, 21 May 830 – 1900 Wednesday, 22 May 830 – 1800
ISAC 2013 Program and Abstracts
19
Committee Meetings during CYTO 2013 All meetings are by invitation only unless specified otherwise. Sunday, 19 May, 2013 ISAC Scholars/Student Travel Awards Breakfast 730 – 830 Room 28D Cytometry Part A Editorial Board Meeting 1230 – 1400
Room 28E
Associated Societies Luncheon 1245 – 1345 Room 28D
Monday, 20 May, 2013 Finance Committee 730 – 830
Room 28C
Certification Advisory Committee 730 – 830 Room 28E Membership Services Committee 730 – 830 Room 28D
Tuesday, 21 May, 2013 Current Protocols Board Meeting 1200 – 1330 Room 28E
Wednesday, 22 May, 2013 Flow Cytometry Data Standards Task Force 730 – 830 Room 28C CYTO 2014 Planning Meeting 730 – 830 Room 28E ISAC Scholar Review Committee 730 – 830 Room 28D ISAC Scholars Luncheon 1200 – 1300 20
Room 28D ISAC 2013 Program and Abstracts
Congress Overview
Congress Overview
Special Lectures
All Congress activities (with the exception of the Closing Reception at the Stingaree) will be held at the San Diego Convention Center. Exhibits/Posters, Cyto Registration and First Aid offices are located on the Ground Level. All other rooms are located on the Upper Level of the Convention Center.
Saturday, 18 May 2013 Introduction to Image-Based Cytometry Course
Room 29C
900 – 1215
Introduction to Flow-Based Cytometry Course
Room 29D
930 – 1600
Advanced Data Analysis Course
1100 – 1800
Scientific Registration Open Lobby
1100 – 1800
Exhibitor Registration Open Lobby
1230 – 1400
Scientific Tutorials 1 – 5
1300 – 1700
CYTO Innovation
1415 – 1545
Scientific Tutorials 6 – 10
Rooms 30 – 33
1600 – 1730
Scientific Tutorials 11 – 15
Rooms 30 – 33
Saturday, 18 May
900 – 1215
Room 29AB Sunday, 19 May
Rooms 30 – 33 Room 28E Monday, 20 May
700 – 1900
Scientific Registration Open Lobby
700 – 1900
Exhibitor Registration Open Lobby
900 – 1030
Opening Remarks:
Wallace H. Coulter Centennial Lecture
1030 – 1100
Coffee Break
1100 – 1230
Parallel Sessions
1245 – 1345
Exceptional Student Award Presentations
1245 – 1345
Commercial Tutorials
1400 – 1530
State-of-the-Art Lectures
1530 – 1545
Coffee Break
1545 – 1715
Workshops
1730 – 1830
Robert Hooke Lecture
1830 – 1930
Opening Reception
Tuesday, 21 May
Sunday, 19 May 2013
Wednesday, 22 May
Ballroom 20D Foyer/Terrace, Upper Level Rooms 30 – 33
Poster Session
Room 28C Rooms 29 – 33 Ballroom 20D
Commercial Tutorials & Exhibits
Foyer/Terrace, Upper Level Rooms 30 – 33 Ballroom 20D Foyer/Terrace, Upper Level
Oral Session Abstracts
Monday, 20 May 2013 Scientific Registration Open Lobby
730 – 1830
Exhibitor Registration Open Lobby
830 – 1000
Frontiers Session 1
1000 – 1015
Coffee Break
1015 – 1145
Parallel Sessions
1130 – 1900
Commercial Exhibits & Poster Viewing
Exhibit Hall GH
1200 – 1330
Exhibitor Showcase
Exhibit Hall GH
Ballroom 20D Foyer/Terrace, Upper Level Rooms 30 – 33
Speaker/Author Index
ISAC 2013 Program and Abstracts
Poster Session Abstracts
730 – 1830
21
Congress Overview Special Lectures Saturday, 18 May
1200– 1330
Lunch for CYTO Attendees
Exhibit Hall GH
1330 – 1500
Plenary Session 1
1500 – 1545
Coffee Break
1545 – 1715
Workshops
1715 – 1845
Poster Session 1
Exhibit Hall GH
1800 – 1900
Happy Hour
Exhibit Hall GH
1900 – 2200
Core Managers Forum
Ballroom 20D Exhibit Hall GH Rooms 30 – 33
Room 29
Wednesday, 22 May
Tuesday, 21 May
Monday, 20 May
Sunday, 19 May
Tuesday, 21 May 2013 700 – 1900
Poster Viewing
Exhibit Hall GH
730 – 1830
Scientific Registration Open Lobby
800 – 1000
Frontiers Session 2
1000 – 1015
Coffee Break
1015 – 1145
Parallel Sessions
1030 – 1830
Exhibitor Registration Open Lobby
1215 – 1315
Presidential Award for Excellence Presentations
1130 – 1900
Commercial Exhibits
Exhibit Hall GH
1215 – 1315
Commercial Tutorials
Rooms 29 – 33
1330 – 1500
Plenary Session 2
1500 – 1545
Coffee Break
1545 – 1715
Workshops
1715 – 1845
Poster Session 2
Exhibit Hall GH
1800 – 1900
Happy Hour
Exhibit Hall GH
Ballroom 20D Foyer/Terrace, Upper Level Rooms 30 – 33 Room 28C
Ballroom 20D Exhibit Hall GH Rooms 30 – 33
Speaker/Author Index
Poster Session Abstracts
Oral Session Abstracts
Commercial Tutorials & Exhibits
Poster Session
Wednesday, 22 May 2013 700 – 1600
Poster Viewing
Exhibit Hall GH
730 – 1700
Scientific Registration Open Lobby
830 – 1000
Frontiers Session 3
1000 – 1015
Coffee Break
1015 – 1145
Parallel Sessions
1030 – 1700
Exhibitor Registration Open Lobby
1130 – 1630
Commercial Exhibits
Exhibit Hall GH
1215 – 1315
Commercial Tutorials
Rooms 29 – 33
1330 – 1500
Plenary Session 3
1500 – 1545
Coffee Break
Exhibit Hall GH
1500 – 1600
Poster Session 3
Exhibit Hall GH
1615 – 1645
ISAC Business Meeting
Ballroom 20D
1645 – 1730
Awards Ceremony
Ballroom 20D
Ballroom 20D Foyer/Terrace, Upper Level Rooms 30 – 33
Ballroom 20D
1900 – 2300 Closing Reception Stingaree (see page 11 for details) 22
ISAC 2013 Program and Abstracts
Congress Overview Special Lectures
Visit the Exhibits & Posters Monday, 20 May 1130 – 1900
Poster Viewing and Commercial Exhibits
1200 – 1330
Exhibitor Showcase
1200 – 1330
Lunch Provided
1500 – 1545
Coffee Break
1715 – 1845
Poster Session 1 (Authors of odd numbered posters present)
1800 – 1900
Happy Hour
Sunday, 19 May
Authors Must Place Posters on Boards Saturday, 18 May
700 – 1100
Poster Viewing
1100 – 1400
Concession open for purchase of lunch/snacks
1130 – 1900
Commercial Exhibits
1500 – 1545
Coffee Break
1715 – 1845
Poster Session 2 (Authors of even numbered posters present)
1800 – 1900
Happy Hour
Tuesday, 21 May
700 – 1900
Monday, 20 May
Tuesday, 21 May
Wednesday, 22 May
Wednesday, 22 May Poster Viewing
1100 – 1400
Concession open for purchase of lunch/snacks
1130 – 1630
Commercial Exhibits
1500 – 1545
Coffee Break
1500 – 1600
Poster Session 3 (all authors present)
Poster Session
700 – 1600
Commercial Tutorials & Exhibits
Posters must be removed by 1630 on Wednesday, 22 May.
Oral Session Abstracts
Don’t forget to exchange your coupon for a ticket to the Closing Reception at the Stingaree!
Poster Session Abstracts
(ticket required for admittance; see page 11 for details)
Speaker/Author Index
ISAC 2013 Program and Abstracts
23
Congress Overview
Special Lectures
Special Lectures
Wallace H. Coulter Centennial Lecture Sunday, 19 May, 900 – 1030 Ballroom 20D
Sunday, 19 May
Saturday, 18 May
Wallace Coulter was a pioneering figure in the field of cytometry. His invention of the Coulter principle for electronic particle sizing revolutionized the counting and sizing of blood cells, and stimulated the design of the first flow cytometers and cell sorters. The Coulter Corporation sold some of the first flow cytometers, and employed many pioneers in the field of cytometry. Wallace Coulter’s legacy lives on through the Wallace H. Coulter Foundation, which actively supports the development and translational application of new biomedical technologies. The Foundation is also a valued partner of ISAC and supporter on issues of mutual interest, including education, data sharing, and certification. ISAC is pleased to recognize this pioneering inventor, entrepreneur, and philanthropist on the 100th anniversary of his birth with the Wallace H. Coulter Centennial Lecture to open CYTO 2013. For more information on Wallace H. Coulter and the Wallace H. Coulter Foundation, visit www.whcf.org.
Monday, 20 May
Spatial Systems Biology, Joe Gray
Wednesday, 22 May
Tuesday, 21 May
The Wallace H. Coulter Centennial Lecture will be presented by Professor Joe Gray, of the Knight Cancer Center and the Oregon Health and Sciences University. Dr. Gray, a pastpresident of ISAC and Fulwyler Award winner, has been a pioneer in the development and translational application of cytometry technologies. He currently leads the OHSU Center for Spatial Systems Biomedicine, which is dedicated to the use of advanced measurement technologies, especially omic and multiscale imaging, to improve medical diagnosis and treatment.
Marylou Ingram Lecture Wednesday, 22 May, 1430 – 1500 Ballroom 20D
Oral Session Abstracts
Commercial Tutorials & Exhibits
Poster Session
Marylou Ingram’s contributions to the field of cytometry span nearly 70 years. Dr. Ingram’s distinguished career in academic medicine and research, includes faculty service at the University of Rochester, Caltech and the University of Miami, at Los Alamos National Laboratory, as a consultant to the National Cancer Institute, the FDA, Brookhaven National Laboratory, NASA and other organizations. She was a pioneer in automated cell analysis, played a key role in developing automated cell analysis systems, and was founding director of the Institute for Cell Analysis at the University of Miami. Dr. Ingram spent two of her sabbaticals working directly with Wallace Coulter in the very early days of automated cell analysis, and she regularly interacted with Wallace Coulter as he developed new technologies for analysis. Since 1982, she has been at the Huntington Medical Research Institute in Pasadena, California, where she is a Senior Research Scientist and heads the Tissue Engineering & In Vitro Systems program, and leads a research program on tumor spheroids with the aim of creating better models for studying tumor growth and drug responsiveness.
Speaker/Author Index
Poster Session Abstracts
The Marylou Ingram Lecture will be presented by Dr. Vera Donnenberg of the McGowan Institute of Regenerative Medicine at the University of Pittsburg. Dr. Donnenberg is an Associate Professor of Cardiothoracic Surgery in the School of Medicine at the University of Pittburgh, where her lab focuses on the application of flow cytometry to cancer biology and regenerative medicine. Dr. Donnenberg serves on the Editorial Board of Cytometry Part A, and is a member of the International Society for Advancement of Cytometry, the American College of Clinical Pharmacology, and American Association for Cancer Research.
24
ISAC 2013 Program and Abstracts
Congress Overview
Daily Program SATURDAY, 18 MAY 2013
Special Lectures
Pre-Congress Courses (separate registration required)
Saturday, 18 May
900 – 1215
Room 29C
Introduction Image-Based Cytometry
900 – 1215
Room 29D
Introduction to Flow-Based Cytometry Sunday, 19 May
930 – 1600
Room 29AB
Advanced Data Analysis Course
Monday, 20 May
Scientific Tutorials
(separate registration required) 1230 – 1400
Tutorials 1 – 5 1230
1
Tuesday, 21 May
Room 33AB
Apoptosis, Autophagy and DNA Damage W. Telford and Z. Darzynkiewicz. NCI, NIH and New York Med. Col.
1230
2
Wednesday, 22 May
Room 32AB
Cytometer Performance Characterization and Standardization R. Hoffman. BD Biosciences, San Jose, CA.
Room 31ABC
1230
3
Poster Session
Approaches in Image-Based High Content Screening S. Heynen-Genel and J.H. Price. Sanford-Burnham Med. Res. Inst., La Jolla and Vala Sciences Inc., San Diego.
Room 30CDE
4
Biosafety: Risk Assessment and SOP Development K. Holmes. NIAID, NIH.
Commercial Tutorials & Exhibits
1230
Room 30AB
1230
5
High Throughput and High Content Screening for Flow Cytometry J.P. Robinson, P. Narayanan and R. Jepras. Purdue Univ., Amgen, Seattle and GSK, Middlesex, U.K.
Oral Session Abstracts
CYTO Innovation
(separate registration required) 1300 – 1700
Poster Session Abstracts
Room 28E
ISAC 2013 Program and Abstracts
Speaker/Author Index
ISAC presents CYTO lnnovation 2013, a forum for discussing the challenges and opportunities for the development and commercialization of cell analysis technologies. The CYTO Innovation Program will include short talks offering perspectives on current trends in the industry, a panel discussion, and a showcase of short presentations by innovators and entrepreneurs on new technologies and applications for cell analysis. If you are involved in the development, evaluation, or commercialization of new cell analysis technologies, plan to join us for this stimulating program. 25
Congress Overview
Scientific Tutorials
(separate registration required) 1415 – 1545
Special Lectures
Tutorials 6 – 10 Room 33AB
6
Saturday, 18 May
1415
Proliferation Tutorial: Cell Cycle Progression and Division – Unraveled by Flow Cytometry K. Price, K.A. Muirhead and P.K. Wallace. Malaghan Inst. of Med. Res., Wellington, NZ, SciGro Inc./MidWest Ofc., Madison, WI and Roswell Park Cancer Inst., Buffalo.
Room 32AB Sunday, 19 May
1415
7
Cell Sorting: Fundamentals, Applications and Troubleshooting G. Osborne. Univ. of Queensland, Australia.
Room 31ABC
Monday, 20 May
1415
8
Image Quantification and Analysis Using CellProfiler D. Logan. Broad Inst. of Harvard and MIT.
Room 30CDE
1415
9
Analysis of Receptor Dynamics Using Flow Cytometry A. Chigaev and Y. Wu. Univ. of New Mexico.
Tuesday, 21 May
Room 30AB
Wednesday, 22 May
1415
10
Evaluation and Purchase of an Analytical Flow Cytometer: Some of the Numerous Factors to Consider R. Zucker and N. Fisher. U.S. EPA, Durham, NC and Univ. of North Carolina at Chapel Hill.
Scientific Tutorials
(separate registration required) 1600 – 1730
Poster Session
Tutorials 11 – 15 Room 33AB
Commercial Tutorials & Exhibits
1600
11
Seeing a More Colorful World: A Guide to Polychromatic Flow Cytometry P. Chattopadhyay. NIAID, NIH.
Room 32AB
Oral Session Abstracts
1600
12
Analysis and Sorting of Rare Cell Populations J.P. McCoy. NHLBI and Ctr. for Human Immunol., NIH.
Room 31ABC
1600
13
Quantitative FRET Microscopy G. Vereb and J. Szollosi. Univ. of Debrecen, Hungary.
Room 30CDE
Poster Session Abstracts
1600
14
Growing a Cytometry Core Facility: Adding Value with Hardware and Education D. Davies and A. Blanco. Cancer Res. UK, London and University Col. Dublin.
Room 30AB
Speaker/Author Index
1600
26
15
Cell Organizer: Building Models of Cell Structure from Microscope Images and Using Them for High-Content Screening and Cell Simulations R. Murphy, G. Johnson and D. Sullivan. Carnegie Mellon Univ.
ISAC 2013 Program and Abstracts
Congress Overview
Sunday, 19 May 2013 Wallace H. Coulter Centennial Lecture
Special Lectures
900 – 1030 Ballroom 20D
Chair: John Nolan Saturday, 18 May
Spatial Systems Biology Joe Gray. Knight Cancer Center and Oregon Health & Sciences University.
Coffee Break 1030 – 1100 Foyer/Terrace, Upper Level
Sunday, 19 May
Concurrent Parallel Sessions 1100 – 1230
Monday, 20 May
Parallel 1: Flow Cytometry Instrumentation: Spectroscopy Room 33AB
Chair: Diether Rechtenwald Cochair: Michael Zorden
1120
21
Simultaneous Analysis of Multiple Fluorescent Proteins and Fluorochromes by a Novel Spectral Flow Cytometer M. Tomura, K. Futamura, N. Nitta M. Kakuta and M. Furuki. Kyoto Univ. Grad. Sch. of Med. and SONY Corp., Tokyo.
1140
22
Quantitative Real Time Single Cell Spectroscopy in Flow J. Nolan, D. Condello and E. Duggan. La Jolla Bioengineering Inst.
1200
23
Fluorescence Lifetime-Dependent Flow Cytometry in the Time-Domain W. Li, G. Vacca, M. Naivar and J. Houston. New Mexico State Univ., Kinetic River Corp., San Jose, CA and DarklingX LLC, Los Alamos.
Poster Session
Expanding the Capabilities of Mass Cytometry S. Tanner, A. Loboda, D. Bandura, V. Baranov and O. Ornatsky. DVS Sciences Inc., Markham, ON.
Wednesday, 22 May
20
Tuesday, 21 May
1100
Parallel 2: Personalized Medicine 1 Commercial Tutorials & Exhibits
Room 32AB
Chair: Virginia Litwin Cochair: Kewal Asosingh
1120
25
Functional Characterization of Lymphoid Subsets in Chronic Myelogenous Leukemia by Mass Cytometry Phospho-Flow Analysis J. De, R. Fernandez, N. Shah and H. Maecker. Stanford Univ. and UCSF Helen Diller Family Comprehen. Cancer Ctr.
1140
26
Maternal BMI Affects Expression Pattern of Cord Blood T- and NK-Cell-Subtypes A. Tárnok , J. Bocsi, C. Blatt, A. Szabó, S. Melzer and I. Dähnert. Univ. of Leipzig and LIFE Leipziger Res. Ctr.
ISAC 2013 Program and Abstracts
Speaker/Author Index
Remarkable Durability of Ag85a-Specific CD4 T Cell Memory Responses Up to 6 Years after Mva85a Vaccination E. Smit, M. Tameris, E.J. Hughes, A. Veldsman, L. van der Merwe, H. Geldenhuys, M. Hatherill, H. McShane, W. Hanekom, H. Mahomed and T. Scriba. Univ. of Cape Town and Univ. of Oxford.
Poster Session Abstracts
24
Oral Session Abstracts
1100
27
Congress Overview Special Lectures
1200
27
Compromised Innate and Adaptive Immune Responses during Yellow Fever Vaccination in Elderly — A Multiparametric Longitudinal FACS Study R.A. Schulz, R. Stark, J.N. Maelzer, C. Domingo-Carrasco, T. Jelinek, K. Juerchott, N. Babel, A.U. Neumann, M. Niedrig and A. Thiel. Charité, Berlin, Robert Koch Inst., Berlin, Berlin Ctr. for Travel and Trop. Med. and Humboldt Univ. of Berlin.
Parallel 3: Flow Cytometry Software Room 31ABC
1100
28
Reproducing Manual Gating of Flow Cytometry Data by Automating Cell Population Identification J. Taghiyar, R. Droumeva, M. Malekesmaeili, G. Finak R. Gottardo and R. Brinkman. British Columbia Cancer Agcy. and Fred Hutchinson Cancer Res. Ctr., Seattle.
1120
29
FlowCAP: Comparison of Automated and Manual Gating of Standardized Lyoplate Flow Cytometry Data G. Finak, J. Ramey, J. Taghiyar, R. Stanton, A. Brandes, P. Qui, J.P. McCoy, D. Hafler, H. Maecker, T. Mossman, R. Scheuermann, R. Brinkman and R. Gottardo. Fred Hutchinson Cancer Res. Ctr., Seattle, British Columbia Cancer Agcy., J Craig Ventner Inst., San Diego, Broad Inst., Cambridge, MA, Univ. of Texas MD Anderson Cancer Ctr., NHLBI, NIH, Yale Univ. Sch. of Med., Stanford Univ. and Univ. of Rochester Med
1140
30
A Computational Method for Creating and Using Pre-defined Gating Sequence Templates to Automatically Gate High-Dimensional Flow Data S. Meehan, C. Meehan, W.A. Moore, D.R. Parks and L.A. Herzenberg. Stanford Univ., Burnaby, BC and Stanford, CA and Univ. of Toronto.
1200
31
Simplicial Analysis in Flow Cytometry Data Processing: Enabling SVM and HDP Cell Classification via Standardization of Cell-Signal Simplices B. Rajwa, F. Akova, A. Pothen and M. Murat Dundar. Purdue Univ. and Indiana Univ.-Purdue Univ. Indianapolis.
Wednesday, 22 May
Tuesday, 21 May
Monday, 20 May
Sunday, 19 May
Saturday, 18 May
Chair: Nima Aghaeepour Cochair: Enrico Lugli
Parallel 4: Image Cytometry Technology Poster Session
Room 30CDE
Chair: Gustavo Rohde Cochair: Bartek Rajwa 32
Next-Generation FLIM: Modulated All Solid-State Camera System I. Young, Q. Zhao, B. Schelen, B. Schelen, R. Schouten, J. Bosiers, R. Leenen, I. Peters and K. Jalink, Delft Univ. of Technol., Teledyne DALSA, Eindhoven, NKI - Dutch Cancer Inst., Amsterdam and Lambert Instruments, Roden, Netherlands.
1120
33
Intravital Marker-Free NAD(P)H Fluorescence Lifetime Imaging – In Vivo Selective Enzyme Detection R. Niesner, A. Mossakowski, J. Pohlan, M. Radbruch, J. BayatSarmadi, F. Paul, A.E. Hauser and H. Radbruch. Charité - Univ. Hosp. and DRFZ, Berlin.
1140
34
Ultra High Throughput Image Cytometry via Continuous Scanning J. Price, G. Gemmen, B. Azimi, M. Guigli and J. Price. Vala Sciences Inc., San Diego and Sanford-Burnham Med. Res. Inst., La Jolla.
1200
35
Automated Intracellular FRET Measurements Using Hyperspectral Microscopy and Feature Extraction S. Leavesley, A. Britain and T. Rich. Univ. of South Alabama.
Speaker/Author Index
Poster Session Abstracts
Oral Session Abstracts
Commercial Tutorials & Exhibits
1100
28
ISAC 2013 Program and Abstracts
Congress Overview
Parallel 5: Multiplexed and Microparticle-based Assays Room 30AB
Amplifying DNA Beads Assay in Flow Cytometry: A Platform Technology Based on Exonuclease III-Aided Target Recycling J. Lu and D. Jin. Macquarie Univ., Australia.
1120
37
Development of a Glycoprofiling Method Using Multiplex Microspheres L. Yang, C. Leoff and R. Woods. Glycosensors and Diagnostics, San Diego, Univ. of Georgia and Glycosensors and Diagnostics, Athens, GA.
1140
38
Multiplexed Microsphere Protease Assays and High Throughput Flow Cytometry for Drug Discovery J. Zhu, L. Sklar, B. Edwards and S.W. Graves. Univ. of New Mexico.
1200
39
Significantly Improve Bead Recovery of Flow Cytometry Assay by Utilising a New Technology Platform for Sample Incubation M. Tenje, N. LeBlanc, M. Evander, B. Hammarström, H. Xia, A. Tojo, S. Belák and T. Laurell. Lund Univ., Natl. Vet. Inst., Uppsala, Swedish Univ. of Agr. Sci. and Dongguk Univ., South Korea.
Sunday, 19 May
36
Saturday, 18 May
1100
Special Lectures
Chair: Marie Iannone Cochair: Yang Wu
Monday, 20 May
Exceptional Student Award Presentations 12:45 – 13:45 Room 28C
Tuesday, 21 May
Award Finalists Ali Vaziri Gohar Determining Intracellular Protein Localization with Fluorescence Lifetime-Based Flow Cytometry
Wednesday, 22 May
Irina Polshchitcina Kinetic Study of Morphological Changes in Human Lymphocytes during Early Stages of Apoptosis using Scanning Flow Cytometry Charles Shields IV Acoustofluidic Cell Sorting via Negative Acoustic Contrast Capture Colloids
Poster Session
Jiangbo Zhao SUPER Dots: The Next-Generation Bio-Labels
Commercial Tutorials Commercial Tutorials & Exhibits
1245 – 1345
Featured Companies
Oral Session Abstracts
Cytek Development – Room 33AB Beckman Coulter Life Sciences – Room 32AB Sony Biotechnology, Inc. – Room 31ABC EMD Millipore – Room 30CDE eBioscience, an Affymetrix company – Room 30AB Beckman Coulter Life Sciences – Room 29CD Fluidigm – Room 29AB
Poster Session Abstracts
See pages 66– 68 for full details.
Speaker/Author Index
ISAC 2013 Program and Abstracts
29
Congress Overview
State-of-the-Art Lectures 1400 – 1530 Ballroom 20D
Sunday, 19 May
Saturday, 18 May
Special Lectures
Chair: J. Paul Robinson Cochair: Andrea Cossarizza 1400
17
Translating Acoustophoretic Cell Handling to Clinical Applications T. Laurell. Lund Univ., Sweden.
1430
18
Signaling Networks Regulating Cellular Growth and Form C. Bakal. Inst. of Cancer Res., London.
1500
19
Discovery of Small Molecules That Control Cell Differentiation P. Bartunek. Inst. of Molec. Genet., Prague.
Coffee Break
Monday, 20 May
1530 – 1545 Foyer/Terrace, Upper Level
Concurrent Workshop Sessions 1545 – 1715
Tuesday, 21 May
Workshop 1 Room 33AB
Wednesday, 22 May
1545
40
Navigating the Labyrinth of Regulated Flow Cytometry in Drug Development V. Litwin, J.J. Stewart, J. Olsen, J. Puchalski, C. Green and C. Wiwi. Covance Inc., Flow Contract Site Lab., Amgen Inc. and Celgene Corp.
Workshop 2 Room 32AB
41
Poster Session
1545
The Delivery of Image-Based Cytometry Education J. Nolan, B. Rajwa, R. Errington, G. Vereb, S.J. Lockett, A. Parwani and G.K. Rohde. La Jolla Bioengineering Inst., Purdue Univ., Cardiff Univ., U.K., Univ. of Debrecen, Hungary, SAIC- Frederick Natl. Lab. and Carnegie Mellon Univ.
Commercial Tutorials & Exhibits
Workshop 3 Room 31ABC
Oral Session Abstracts
1545
42
Biosafety: Biosafety Policy Meets Real Life Scenarios K. Holmes and S.P. Perfetto. NIAID, NIH.
Workshop 4 Room 30CDE
Poster Session Abstracts
1545
43
Quantitative Cytometry — Calibration and Standardization L. Wang and R. Hoffman. NIST, Gaithersburg, MD and BD Biosciences, San Jose, CA.
Workshop 5
Speaker/Author Index
Room 30AB
1545
30
44
Functional Analysis of Mitochondria and Transporters G. Babcock and P Narayanan. Univ. of Cincinnati and Amgem
ISAC 2013 Program and Abstracts
Congress Overview
Robert Hooke Lecture 1730 – 1830 Ballroom 20D
Special Lectures
Chair: Larry Sklar TBA
Saturday, 18 May
Opening Reception 1830 – 1930 Foyer/Terrace, Upper Level
Sunday, 19 May
Monday, 20 May 2013
Monday, 20 May
Frontiers Session 1: Innovation 830 – 1000
Ballroom 20D
46
Use of Kinetic Imaging Cytometry to Develop Pharmacological Approaches to Cardiac Regeneration and Preservation of Contractile Function M. Mercola. SanfordBurnham Med. Res. Inst., and UCSD, La Jolla.
915
47
Non-genetic Cell Population Heterogeneity: Implications for Cell Differentiation and Cancer Progression S. Huang. Inst. for Systs. Biol., Seattle.
Wednesday, 22 May
830
Tuesday, 21 May
Chair: Jeff Price Cochair: William Telford
Coffee Break
Poster Session
1000 – 1015 Foyer/Terrace, Upper Level
Concurrent Parallel Sessions Commercial Tutorials & Exhibits
1015 – 1145
Parallel 6: Cell Proliferation and Death Room 33AB
Live Cell Analysis of NCAM Polysialylation in Micro-Communities Using the Novel Combination of an Antibody-Mimetic EGFP-Endosialidase and the Viability Dye DRAQ7 P. Smith, M. Wiltshire, S. Chappell, L. Patterson, S. Shnyder, R. Falconer and R. Errington. Sch. of Med., Cardiff Univ. and Univ. of Bradford, U.K.
1035
49
Cytometric Assessment of DNA Damage- and mTOR-Signaling, the Factors Contributing to Aging and Senescence. Z. Darzynkiewicz, H. Zhao and D. Halicka. New York Med. Col.
ISAC 2013 Program and Abstracts
Speaker/Author Index
48
Poster Session Abstracts
1015
Oral Session Abstracts
Chair: Rachel Errington Cochair: Katarzyna Piwocka
31
Congress Overview Special Lectures
1055
50
Dissecting the Intricate Network of the Cell Death Machinery: Simultaneous Detection of Apoptosis and Autophagy Using Flow Cytometry R. Pal, Y. OheneAbuakwa, T. Rutherford and F. Gibson. St George’s Univ. of London.
1115
51
Development and Qualification of a Whole Blood Assay to Monitor pHH3 and Cell Cycle in Patients with Acute Myeloid Leukemia Treated with Aurora Kinase Inhibitors C. Green, C. Ma, J. Ferbas and G. Juan. Amgen, Ventura and Thousand Oaks, CA.
Saturday, 18 May
Parallel 7: Image Cytometry Applications Room 32AB
Wednesday, 22 May
Tuesday, 21 May
Monday, 20 May
Sunday, 19 May
Chair: Stephen Lockett Cochair: Anis Larbi 1015
52
Understanding Subcellular Filament Networks from Fluorescence Microscopy Images G. Rohde, S. Basu, J. Li, K. Dahl and R. Murphy. Carnegie Mellon Univ.
1035
53
New Approaches to Study Neuronal Connectivity in a High-Content Format N. Prigozhina, J. Seldeen, F. Cerignoli, R. Basa, J. Price and P. McDonough. Vala Sciences Inc., San Diego.
1055
54
Addressing Uncertainty in the Automated Evaluation of Stem Cell Colony Quality M. Halter, Y-S. Li-Baboud, A. Peskin, P. Bajcsy, D. Hoeppner and A.L. Plant. NIST, Gaithersburg, MD and Leber Inst. for Brain Develop., Baltimore.
1115
55
A Non-parametric Method for the Automated Discovery of Perturbagen Responses in High-Content Analysis G. Johnson, J. Kangas, A. Dovzhenko, K. Voigt, S. Bhavani, K. Palme and R. Murphy. Carnegie Mellon Univ. and Albert Ludwigs Univ. Freiburg, Germany.
Parallel 8: Flow Cytometry Instrumentation Room 31ABC
1015
56
Accelerated Cell Sorting Using In-Line Sample Pre-enrichment B. Warner, L. Yu, J. Trotter, M. Jaimes, M. Blom, W. Buesink, A. Lenshof, T. Laurell and T. Lau BD Biosciences, San Jose, CA, Micronit Microfluidics, Netherlands and Lund Univ., Sweden.
1035
57
Microflow1, a Portable Flow Cytometer for Space Travel: In Preparation for the International Space Station Demonstration C. Riviere, O. Mermut, G. DubeauLaramee and L. Cohen. INO, Quebec City and Canadian Space Agcy., Saint-Hubert, QC.
1055
58
An Extremely Parallel Flow Cytometer for Rapid Cellular Analysis S. Graves, P.P. Austin Suthanthiraraj, M.E. Piyeasena and A. Shreve. Univ. of New Mexico.
1115
59
Acoustofluidic Cell Sorting via Negative Acoustic Contrast Capture Colloids C. Shields IV, L. Johnson, L. Gao and G. Lopez. Duke Univ. and Triangle MRSEC, Durham, NC.
Speaker/Author Index
Poster Session Abstracts
Oral Session Abstracts
Commercial Tutorials & Exhibits
Poster Session
Chair: Geoffrey Osborne Cochair: Sung Hwan Cho
32
ISAC 2013 Program and Abstracts
Congress Overview
Parallel 9: Personalized Medicine 2 Room 30CDE
1035
61
Immune Monitoring of Human Kidney Allografts with Biopsy Multicolor Flow Cytometry and Cytokines K. Muczynski, N. Leca and S. Anderson. Univ. of Washington.
1055
62
Metastatic Breast Cancer Stem/Progenitor Populations Survive Consolidation Chemotherapy, Circulate and Disseminate to Bone Marrow A. Donnenberg, V.S. Donnenberg, R. Landreneau and A. Brufsky. Univ. of Pittsburgh Sch. of Med.
1115
63
Relapsing-Remitting MS Patients Have Significant Differences in Circulating B Cells Subset and Phenotype Compared with Healthy Controls, and IFN&[beta]-1b Treatment Can Alter the Composition and Phenotype of These Subsets D. Mielcarz, J. DeLong, A. Bergeron, K. Smith, A. Heyn, L. Kasper and J. Channon. Geisel Sch. of Med. at Dartmouth.
Monday, 20 May
Complex Changes in Invariant Natural Killer T Cells in Patients with Different Clinical Forms and Treatments of Multiple Sclerosis S. De Biasi, M. Nasi, A.M. Simone, D. Ferraro, F.F. Vitetta, L. Gibellini, M. Pinti, A. Del Giovane, P. Sola and A. Cossarizza. Univ. of Modena and Reggio Emilia and Nuovo Ospedale Civile Sant’Agostino Estense (NOCSAE), Italy.
Sunday, 19 May
60
Saturday, 18 May
1015
Special Lectures
Chair: Cherie Green Cochair: Padma Narayanan
Tuesday, 21 May
Parallel 10: Cellular Assay Systems Room 30AB
1035
65
Analysis and Sorting of Antigen-Specific Antibody Secreting Cells in Mixed Cultures Using the Affinity Matrix Technology A. Taddeo, H-D. Chang, V. Gerl, B. Hoyer, A. Radbruch and F. Hiepe. DRFZ and Charité - Med. Univ. Berlin, Campus Mitte.
1055
66
Developing Measures of Cellular Potency for Therapeutic Transplantation D. Kaplan, H.M. Lazarus and N.M. Kaye. Pathfinder Biotech, Cleveland and Case Western Reserve Univ.
1115
67
Sorting of Specific Lymphocyte Populations from Peripheral Blood Progenitor Cell Products Using a Novel Micro-Chip Based Acoustophoretic Platform A. Urbansky, A. Lenshof, A. Jamal, J. Dykes, I. Åstrand-Grundström, T. Laurell and S. Scheding. Lund Univ., Sweden.
Oral Session Abstracts
A High-Throughput Method for Creating Uniform 3D Tissue Models J. De Lora, D. Kalb, A. Martinic, A. Trujillo, T. Woods, A. Shreve and J. Freyer. Univ. of New Mexico.
Commercial Tutorials & Exhibits
64
Poster Session
1015
Wednesday, 22 May
Chair: Tim Bushnell Cochair: Alfonso Blanco-Fernandez
Commercial Exhibits
Poster Session Abstracts
1130 – 1900 Exhibit Hall GH
See pages 76– 89 for full details.
Speaker/Author Index
ISAC 2013 Program and Abstracts
33
Congress Overview
Poster Viewing
Special Lectures
Exhibitor Showcase
1130 – 1900 Exhibit Hall GH
1200 – 1330 Exhibit Hall GH
Saturday, 18 May
1215 BD Biosciences 1235 handyem 1255 NewSouth Innovations 1315 BioStatus
Sunday, 19 May
Lunch for CYTO Attendees 1200 – 1300
Exhibit Hall GH Monday, 20 May
Plenary Session 1: Immunology 1330 – 1500 Ballroom 20D
Wednesday, 22 May
Tuesday, 21 May
Chair: Andreas Radbruch Cochair: Pratip Chattopadhyay 1330
68
Image Cytometry of Cell Adhesion K. Ley. La Jolla Inst. for Allergy & Immunol.
1400
69
Single-Cell Approaches to Investigate the Immune Response M. Cahalan. Univ. of California, Irvine.
1430
70
Immune Profiles in the Central Nervous System: What We Know, What We Need to Know, and What It Means N. Monson. Univ. of Texas Southwestern Med. Ctr.
Poster Session
Coffee Break 1500 – 1545 Exhibit Hall GH
Commercial Tutorials & Exhibits
Concurrent Workshop Sessions 1545 – 1715
Workshop 6
Oral Session Abstracts
Room 33AB
Poster Session Abstracts
1545
71
Integrating Standards at the Interface of Flow and Image Cytometry H. Minderman, A. Filby, A. Plant, M. Halter, P. Narayanan and A. White. Roswell Park Cancer Inst., Buffalo, Cancer Res. UK, London, NIST, Gaithersburg, MD, Amgen, Seattle and Cincinnati Children’s Hosp.
Workshop 7 Room 32AB
Speaker/Author Index
1545
34
72
Writing, Publishing and Reviewing: Advice and Tips from Cytometry A A. Tarnok, R. Brinkman, V. Donnenberg, H. Ulrich and S. Vice. Univ. of Leipzig , British Columbia Cancer Agcy., Univ of Pittsburgh Hillman Cancer Ctr., Univ of São Paulo and Wiley. ISAC 2013 Program and Abstracts
Congress Overview
Workshop 8 Room 31ABC
1545
73
Special Lectures
High Throughput Flow Cytometry in Pharma B. Edwards and R. Jepras. Univ. of New Mexico and GlaxoSmithKline, Stevenage, U.K.
Workshop 9 Room 30CDE
74
Mesenchymal Stem Cell Identification and Characterization V. Donnenberg, K. Wonnacott, A.D. Donnenberg, H. Ulrich and A. Plant. Univ of Pittsburgh Hillman Cancer Ctr., OD, NIH, Univ. of São Paulo and NIST, Gaithersburg, MD.
Saturday, 18 May
1545
Workshop 10 1545
75
Sunday, 19 May
Room 30AB
Malaria Cytometry H. Shapiro and G. Chojnowski. Howard M. Shapiro, MD, PC and Queensland Inst. of Med. Res., Australia.
Monday, 20 May
Poster Session 1 1715 – 1845 Exhibit Hall GH
Authors of odd numbered boards will present. Tuesday, 21 May
Happy Hour
Wednesday, 22 May
1800 – 1900 Exhibit Hall GH
Core Managers Forum 1900 – 2200 Room 29
Poster Session
This year the Core Managers Forum will have the theme “How can ISAC best serve the Shared Resource Laboratory and its staff”. As facility staff, we know there can be issues with being able to attend CYTO meetings, accessing ISAC member benefits, developing a career track and developing the skill set needed to run or work in a successful cytometry core. How can ISAC help? What are the programs or added value benefits that would aid life in the core? How should they be implemented? There will be two short presentations - Derek Davies (Cancer Research UK, London) “The ISAC SRL Task Force” and Peter Lopez (School of Medicine, New York) “Synergy between ISAC and the Association of Biomolecular Resource Facilities” - but this is your Forum and we want your input so the majority of the evening will be devoted to open discussion. Refreshments will be provided.
Commercial Tutorials & Exhibits Oral Session Abstracts Poster Session Abstracts Speaker/Author Index
ISAC 2013 Program and Abstracts
35
Congress Overview
Tuesday, 21 May 2013
Special Lectures
Poster Viewing 700 – 1900 Exhibit Hall GH
Saturday, 18 May
Frontiers Session 2: Discovery 830 – 1000 Ballroom 20D
Monday, 20 May
Sunday, 19 May
Chair: Ryan Brinkman Cochair: Donat Alpar 830
76
Insights into the Immune System via Single Cell Network Profiling: Towards Improved Disease Classification and Therapeutic Selection A. Cesano. Nodality Inc., South San Francisco.
915
342
Cell-Based Assays in Drug Discovery: Changing the HTS Paradigm H. Djaballah. Mem. Sloan-Kettering Cancer Ctr.
Coffee Break
Tuesday, 21 May
1000 – 1015 Foyer/Terrace, Upper Level
Concurrent Parallel Sessions
Wednesday, 22 May
1015 – 1145
Parallel 11: Image Cytometry Room 33AB
1015
77
Multiparameter Image Cytometry Using Surface Enhanced Raman Scattering Tags and Spectral Imaging E. Liu, E. Duggan and J. Nolan. La Jolla Bioengineering Inst.
1035
78
Phenotyping TILs in Situ: Tissue Cytometric Enumeration of Intra- and ExtraFollicular Foxp3+ Regulatory T Cells in Follicular Lymphoma J. Mansfield, C. van der Loos, L.S. Nelson, C. Rose, H.E. Sandison S. Usher, J.A. Radford, K.M. Linton and R. Byers. PerkinElmer, Hopkinton, MA, Amsterdam Med. Ctr. and Univ. of Manchester.
1055
79
Biophotonic Nanoswitches – Light-Activation of the BH3 Pathway in Cellular Systems R. Errington, R. Mart, S. Chappell, C. Watkins, M. Wiltshire, A. Jones, P. Smith and R. Allemann. Schs. of Med., Chem. and Pharm. and Pharmaceut. Sci., Cardiff Univ., U.K.
1115
80
Quantitative Co-imaging of Activated Signaling Proteins and Downstream Individual Transcripts in Breast Cancer Single Cells S. Kwon, M. Nederlof, K. Chin and J. Gray. Oregon Hlth. & Sci. Univ. and Quantitative Imaging Systs. Inc., Pittsburgh.
Speaker/Author Index
Poster Session Abstracts
Oral Session Abstracts
Commercial Tutorials & Exhibits
Poster Session
Chair: Silas Leavesley Cochair: Anis Larbi
36
ISAC 2013 Program and Abstracts
Congress Overview
Parallel 12: Immune Monitoring Room 32AB
82
The Cytokine Secretion Assay Reveals an Analog to Digital Switch in Cytokine Expression H-D. Chang, J. Koeck, F. Hatam, M. Bardua, S. Kreher, H. Bendfeldt, R. Baumgrass and A. Radbruch. DRFZ, Berlin.
1055
83
Determination of Antigen Localization and Trafficking When Targeted to C-Type Lectin Receptors Using Human Antibody-Antigen Fusion Protein Constructs Harboring Specificity to CD205 or CD206 J. Tario, Jr., T. Keler and P.K. Wallace. Roswell Park Cancer Inst., Buffalo and Celldex Therapeut., Needham, MA.
1115
84
Dissection of Anti-CTLA4-Induced Cytotoxic T Cell Responses in Melanoma P. Kvistborg, D. Philips, S. Kelderman, B. Hemskerk, C. Ottensmeier, D. Speiser, C. Blank, J. Haanen, and T. Schumacher. Netherlands Cancer Inst., Amsterdam, Southampton Univ. Hosp., U.K. and Ludwig Cancer Ctr., Lausanne.
Tuesday, 21 May
1035
Monday, 20 May
Standardization of Whole Blood Immune Phenotype Monitoring for Clinical Trials: Panels and Methods from “The ONE Study” M. Streitz, T. Miloud, M. Kapinsy, M. Reed, E.K. Geissler, J. Hutchinson, K. Vogt, S. Schlickeiser, A. Kverneland, C. Meisel H-D. Volk and B. Sawitzki. Charité - Med. Univ. Berlin, Immunotech S.A.S., Marseille, Beckman Coulter GmbH, Krefeld, Beckman Coulter Inc., Miami and Univ. Hosp. Regensburg, Germany.
Sunday, 19 May
81
Saturday, 18 May
1015
Special Lectures
Chair: Mario Roederer Cochair: Gergely Toldi
Parallel 13: Cell-Derived Microvesicles Room 31ABC
Wednesday, 22 May
Chair: Mehrnoosh Abshari Cochair: Phillip Hexley Flow Cytometric Analysis of Single Lipid Membrane Vesicles S. Stoner and J. Nolan. La Jolla Bioengineering Inst.
1035
86
Novel Approach for Characterizing Circulating Microparticles Using Imaging Flow Cytometry J. Lannigan, C. Rudy and U. Erdbrugger. Univ. of Virginia.
1055
87
Study of Potential Markers for Tumor-Derived Mp in an in Vitro Model of Spiked Whole Blood P. Poncelet, J. Bez, T. Bouriche, and W. Ruf. BioCytex, Marseille and The Scripps Res. Inst.
1115
88
Extracellular Vesicles from Plasma of Healthy Donors, Characterized by CryoElectron Microscopy, Receptor-Specific Gold Labeling and Flow Cytometry A. Brisson, N. Arraud, R. Linares, S. Tan and C. Gounou. Univ. of Bordeaux.
Commercial Tutorials & Exhibits
85
Poster Session
1015
Oral Session Abstracts
Parallel 14: Flow Cytometry Instrument and Software Advances Room 30CDE
1015
89
Poster Session Abstracts
Chair: Bruce Edwards Cochair: Michael Zordan High Throughput Drug Screening J.P. Robinson, V. Patsekin, P.K. Narayanan and N. Li. Purdue Univ. and Amgen, Seattle.
Speaker/Author Index
ISAC 2013 Program and Abstracts
37
Special Lectures
‘Deep Blue’ Lasers for Detecting Low-Level Cyan Fluorescent Protein Expression: An Example of Optimizing Excitation Conditions for Maximum Probe Sensitivity W. Telford, J. Hu-Li, V. Kapoor, N. Hawk, B. Mester, A. Schmidt, R. Kyle, K. Price and G. Le Gros. NCI and NIAID, NIH and Malaghan Inst. of Med. Res., Wellington, New Zealand.
1055
91
Saturday, 18 May
Congress Overview
90
A New Computational Method for Predicting Optimal Reagent-Dye Combinations in Staining Panel Design for High-Dimensional Flow Cytometry W.A. Moore, S.W. Meehan, A.B. Kantor, D.R. Parks and L.A. Herzenberg. Stanford Univ., Stanford, CA and Burnaby, BC.
1115
92
Quantifying the Relationship between Antibody Bound and Signal Observed for a Large Panel of Dye-Conjugated Antibodies Using Complementary Binding by Antibody Capture Beads and Use of This Information in Cytogenie Autocolor to Predict Optimal Multi-color D. Parks, A.B. Kantor, W.A. Moore, S.W. Meehan and L.A. Herzenberg. Stanford Univ., Stanford, CA and Burnaby, BC.
Sunday, 19 May
1035
Commercial Exhibits Monday, 20 May
1130 – 1900 Exhibit Hall GH
See pages 76– 89 for full details.
Tuesday, 21 May
President’s Award for Excellence Presentations Room 28C 12:15 – 13:15
Wednesday, 22 May
Award Finalists Nicolas Arraud Kinetics of Annexin A5 Interaction with Model Membranes, determined by Flow Cytometry.
Poster Session
Silas Leavesley Automated Intracellular Fret Measurements using Hyperspectral Microscopy and Feature Extraction Er Liu Multiparameter Image Cytometry using Surface Enhanced Raman Scattering Tags and Spectral Imaging
Commercial Tutorials & Exhibits
Adriano Taddeo Analysis and Sorting of Antigen-Specific Antibody Secreting Cells in Mixed Cultures using the Affinity Matrix Technology
Commercial Tutorials
Oral Session Abstracts
1215 – 1315
Featured Companies
Speaker/Author Index
Poster Session Abstracts
DVS Sciences – Room 33AB Union Biometrica, Inc. – Room 32AB BD Biosciences – Room 31ABC Sony Biotechnology, Inc. – Room 30CDE Tree Star, Inc. – Room 30AB Miltenyi Biotec GmbH – Room 29CD Bio-Rad – Room 29AB See pages 69 – 71 for full details. 38
ISAC 2013 Program and Abstracts
Congress Overview
Plenary Session 2: Cancer 1330 – 1500 Ballroom 20D
DNA Sequencing Detects Residual Leukemia B. Wood. Univ. of Washington.
1400
94
Quantitative IF – A Molecular Tool for Assay Development and Tissue Quality Assessment in Cancer V. Neumeister, K. Schalper, A. England, E. Zarella, F. Parisi, Y. Kluger, D. Hicks and D. Rimm. Yale Univ. Sch. of Med. and Univ. of Rochester Sch. of Med.
1430
95
Tissue Factor Bearing Microparticles Measured by Impedance-Based Flow Cytometry Predict Thrombosis in Cancer Patients J. Zwicker. Beth Israel Deaconess/ Harvard Med. Sch.
Sunday, 19 May
93
Saturday, 18 May
1330
Special Lectures
Chair: Paul Wallace Cochair: Zbingiew Darzekewicz
Coffee Break Monday, 20 May
1500 -1545 Exhibit Hall GH
Concurrent Workshop Sessions Tuesday, 21 May
1545 – 1715
Workshop 11 Room 33AB
96
Design and Application of Receptor Occupancy Assays Used to Measure Pharmacodynamic Response to Treatment with Biologic Therapies V. Litwin, C. Green, M. Williams, D. Wunderlich, M. Liang and J. Ferbas. Covance Inc., Indianapolis, Amgen Inc., Los Angeles, Genentech, Pfizer Inc. and MedImmune.
Wednesday, 22 May
1545
Poster Session
Workshop 12 Room 32AB
1545
97
Microvesicle Analysis N. Fisher and J. Lannigan. Univ. of North Carolina and Univ. of Virginia.
Commercial Tutorials & Exhibits
Workshop 13 Room 31ABC
1545
98
Oral Session Abstracts
Spectral Imaging and Tissue Cytometry S. Leavesley and J. Mansfield. Univ. of South Alabama and PerkinElmer, Hopkinton, MA.
Workshop 14 Room 30CDE
99
Poster Session Abstracts
1545
Career Development: Cytometry Still Needs You, but Do You Need Cytometry? R. Walker and A. Filby. Babraham Inst. and Cancer Res. UK.
Speaker/Author Index
ISAC 2013 Program and Abstracts
39
Congress Overview
Workshop 15 Room 30AB
Special Lectures
1545
100
Trends in Cytometry Instrumentation S. Graves and G. Vacca. Univ. of New Mexico and Kinetic River Corp., San Francisco.
POSTER SESSION 2
Saturday, 18 May
1715 – 1845 Exhibit Hall GH
Authors of even numbered boards present.
Happy Hour
Speaker/Author Index
Poster Session Abstracts
Oral Session Abstracts
Commercial Tutorials & Exhibits
Poster Session
Wednesday, 22 May
Tuesday, 21 May
Monday, 20 May
Sunday, 19 May
1800 – 1900 Exhibit Hall GH
40
ISAC 2013 Program and Abstracts
Congress Overview
Wednesday, 22 May 2013
Special Lectures
Poster Viewing 700 – 1600 Exhibit Hall GH
Saturday, 18 May
Frontiers Session 3: Translation 830 – 1000 Ballroom 20D
830
101
In Pursuit of Immune Tolerance G. Nepom. Immune Tolerance Network, Seattle.
915
102
Integrating Flow Cytometry and Transcriptomics M. Roederer. NIAID, NIH.
Sunday, 19 May
Chair: Gergely Toldi Cochair: Tomas Kalina
Monday, 20 May
Coffee Break 1000 – 1015 Foyer/Terrace, Upper Level
Tuesday, 21 May
Concurrent Parallel Sessions 1015 – 1145
Parallel 15: Flow Cytometry and Sorting
Wednesday, 22 May
Room 33AB
Chair: Geoffrey Osborne Cochair: Jessica Houston
1035
104
Hollow Core Photonic Crystal Fiber Laser Sources: Closing in on True Tunable Laser Sources for Flow Cytometry W. Telford, V. Kapoor, N. Hawk, Y. Wang, F. Gerome and F. Benabid. NCI, NIH and XLIM Res. Inst., Limoges.
1055
105
Determining Intracellular Protein Localization with Fluorescence Lifetime-Based Flow Cytometry A. Vaziri Gohar, R. Cao, W. Li, P. Jenkins, J.P. Houston and K.D. Houston. New Mexico State Univ.
1115
106
A Label-Free Shape-Based Detection of Activated Platelets with Scanning Flow Cytometry A. Moskalensky, M. Yurkin, A. Konokhova, D. Strokotov, V. Nekrasov, A. Chernyshev and V. Maltsev. Inst. of Chem. Kinet. and Combustion, SB RAS and Novosibirsk State Univ., Russia.
Oral Session Abstracts
A UV-C LED Sheath Fluid Desinfection Module for Flow Cytometric Cell Sorting T. Kaiser, J. Kirsch, J. Glaab, T. Kolbe, M. Kneissl and H-D. Chang. DRFZ, Berlin, Ferdinand Braun Inst., Berlin and Tech Univ. Berlin.
Commercial Tutorials & Exhibits
103
Poster Session
1015
Poster Session Abstracts Speaker/Author Index
ISAC 2013 Program and Abstracts
41
Congress Overview
Parallel 16: Ligand-Receptor Dynamics Room 32AB
Monday, 20 May
Sunday, 19 May
Saturday, 18 May
Special Lectures
Chair: Steve Graves Cochair: Bruno Paredes 1015
107
Kinetics of Annexin A5 Interaction with Model Membranes, Determined by Flow Cytometry N. Arraud, C. Gounou and A. Brisson. Univ. of Bordeaux.
1035
108
Novel Flow Cytometry Assay for Real-Time Detection of Molecular Extension of Lymphocyte Function-Associated Antigen-1 A. Chigaev, Y. Smagley, S. Zhang, M. Haynes, W. Wang and L. Sklar. Univ. of New Mexico.
1055
109
Discovery of Regulators of Receptor Internalization by High Throughput Flow Cytometry Y. Wu, P. Tapia, G. Fisher, A. Waggoner, J. Jarvik and L. Sklar. Univ. of New Mexico and Carnegie Mellon Univ.
1115
110
Studies of Immunological Synapse Formation and Downstream Signaling Events Using the Flowsight and ImageStream Imaging Flow Cytometers H. Pugsley, S. Friend, R. Kong, B. Hall, S. Vaidyanathan and D. Basiji. Amnis of EMD Millipore, Seattle.
Parallel 17: New Probes and Assays Room 31ABC
Commercial Tutorials & Exhibits
Poster Session
Wednesday, 22 May
Tuesday, 21 May
Chair: Joanne Lannigan Cochair: Rachael Walker 1015
111
Development of Brighter Surface Enhanced Raman Scattering Tags for Multiplexed Cytometry J. Nolan and E. Duggan. La Jolla Bioengineering Inst.
1035
112
SUPER Dots: The Next-Generation Bio-Labels. J. Zhao and D. Jin. Macquarie Univ., Australia.
1055
113
RNA Flow Cytometry for Multiplex Gene Expression Analysis for Specific Intracellular mRNAs in Individual Cells E. Park, W. Lomas, M.E. Hanley, D. Mittar, N. Su, Y. Luo and V. Maino. BD Biosciences, San Jose, CA and Adv. Cell Diagnostics Inc., Haywood, CA.
1115
114
Cytometry of Low-Cell-Count Samples: Chipcytometry for Deep Immunophenotyping of Cerebrospinal Fluid and Bronchoalveolar Lavage Cells C. Hennig, A. Mirenska, M. Stangel and G. Hansen. Hannover Med. Sch., Germany.
Parallel 18: Hematological Disorders Room 30CDE
Speaker/Author Index
Poster Session Abstracts
Oral Session Abstracts
Chair: Zofia Maciorowski Cochair: Derek Davies
42
1015
115
Image Cytometry-Based Detection of Aneuploidy by FISH-IS O. Maguire, K. Humphrey, E. Wang, A. Block, S. Sait, P. Wallace and H. Minderman. Roswell Park Cancer Inst., Buffalo.
1035
116
Pilot Investigation of EuroFlow Standardized 8-Color Panel on Different Flow Cytometry Platforms T. Kalina, M. Nováková, M. Vlková, D. Thürner, E. Mejstrikova, Q. Lecrevisse and O. Hrusak. Charles Univ. Prague 2nd Med. Fac. and St. Anne’s Univ. Hosp. and Fac. of Med., Masaryk Univ., Czech Republic and Univ. of Salamanca, Spain.
ISAC 2013 Program and Abstracts
Use of Imaging Flow Cytometry as an Assay for Sickling Capacity in Patients with Sickle Cell Anemia L. Samsel, E. van Beers, L. Mendelsohn, R. Saiyed, P. McCoy and G. Kato. NHLBI, NIH.
1115
118
Kinetic Study of Morphological Changes in Human Lymphocytes during Early Stages of Apoptosis Using Scanning Flow Cytometry I. Polshchitcina, D. Strokotov and V. Maltsev. Inst. of Chem. Kinet. and Combustion, SB RAS and Novosibirsk State Univ., Russia.
Special Lectures
117
Congress Overview
1055
Saturday, 18 May
Commercial Exhibits 1130 – 1630 Exhibit Hall GH
Sunday, 19 May
See pages 76– 89 for full details.
Commercial Tutorials 1215 – 1315
Monday, 20 May
Featured Companies
Tuesday, 21 May
EMD Millipore – Room 33AB Molecular Devices LLC – Room 32AB BD Biosciences – Room 31ABC PARTEC – Room 30CDE Verity Software House – Room 30AB Life Technologies – Room 29CD De Novo Software – Room 29AB
Wednesday, 22 May
See pages 72 – 74 for full details.
Plenary Session 3: Stem Cells 1330 – 1500 Ballroom 20D
13:30 119
Poster Session
Chair: Paul Smith Cochair: Anne Plant
120
Identification and Targeting of Leukemia Stem Cells M. Guzman. Weill Cornell Med. Col.
1430
121
The Marylou Ingram Lecture: Genomic and Phenotypic Pedigree of Breast Cancer Cell Subsets V. Donnenberg, J. Hicks and A. Donnenberg. Univ of Pittsburgh Hillman Cancer Ctr.
Oral Session Abstracts
1400
Commercial Tutorials & Exhibits
Neural Stem and Progenitor Cells in Human Cortical Development and Evolution A. Kriegstein, J. Lui and D. Hansen. UCSF.
Coffee Break
Poster Session Abstracts
1500 – 1545 Exhibit Hall GH
Speaker/Author Index
ISAC 2013 Program and Abstracts
43
Congress Overview
POSTER SESSION 3 1500 – 1600 Exhibit Hall GH
Special Lectures
All authors present. Authors must remove their posters from boards from 1600 – 1630.
Saturday, 18 May
ISAC BUSINESS MEETING 1615 – 1645 Ballroom 20D
Awards Ceremony Sunday, 19 May
1645 – 1730 Ballroom 20D
Master of Ceremonies: Paul J. Smith, Awards Committee Chair and Past President Monday, 20 May
Recognition of New ISAC Scholars
Tuesday, 21 May
Michael Halter Er Liu Yiqing Lu Frank Alexander Schildberg Joseph Tario, Jr.
Cytometry Part A: 2012 Best Paper Award
Wednesday, 22 May
Single-Cell Mass Cytometry Adapted to Measurements of the Cell Cycle G.K. Behbehani, S.C. Bendall, M.R. Clutter, W.J. Fanti, G.P. Nolah
Exceptional Student Award Finalists
Poster Session
Ali Vaziri Gohar Irina Polshchitcina Charles Shields IV Jiangbo Zhao
Commercial Tutorials & Exhibits
Nicolas Arraud Silas Leavesley Er Liu Adriano Taddeo
Oral Session Abstracts
President’s Award for Excellence Finalists
To Be Announced:
Poster Session Abstracts
Distinguished Service Award Membership Award Outstanding Poster Awards The Fulwyler Award for Innovation Excellence
Speaker/Author Index
Closing Reception at the Stingaree 1900 – 2300
Tickets required for admittance. See page 11 for full details. 44
ISAC 2013 Program and Abstracts
Congress Overview
Multimedia and Poster Sessions Exhibit Hall GH
Special Lectures
Author presentation and discussion times:
Tuesday, 21 May 700 – 1900 1715 – 1845
Poster Viewing Poster Session 2: Authors of EVEN numbered poster boards present
Sunday, 19 May
Authors must set up posters on assigned board Poster Viewing Poster Session 1: Authors of ODD numbered poster boards present
Saturday, 18 May
Monday, 20 May 700 – 1100 1130 – 1900 1715 – 1845
Monday, 20 May
Wednesday, 22 May 700 – 1600 Poster Viewing 1500 – 1600 Poster Session 3: All authors present 1600 – 1630 All posters must be removed from the boards.
Multimedia Presentations
B2
123
Study of Sensitivity and Specificity of DNA Image Cytometry in Cervical Squamous Lesions X. Sun, H. Li and M. Zhang. Wuhan Landing Med. High-Tech Co. Ltd. and Hubei Zhong Shan Hosp., Wuhan, China.
B3
124
Simultaneous Recording of Action Potentials and Calcium Transients from Stem Cell-Derived Cardiomyocytes: Applications for Cardiotoxicity Testing R. Whittaker, R. Vega, R. Ingermanson, F. Cerignoli, R. Towart, D. Gallacher, M. Mercola and J.H. Price. Vala Sciences Inc., San Diego, Ctr. of Excellence for Cardiovasc. Safety Res., Beerse, Belgium, Sanford-Burnham Med.Res. Inst., La Jolla and UCSD.
Commercial Tutorials & Exhibits
Population-Based Study of Automated DNA Image Cytometry as a Screening Method for Cervical Cancer in Rural Areas of China X. Sun, H. Li and M. Zhang. Wuhan Landing Med. High-Tech Co. Ltd. and Hubei Zhong Shan Hosp., Wuhan, China.
Poster Session
122
Wednesday, 22 May
B1
Tuesday, 21 May
Automated Microscopy
Cell Proliferation and Death B4
125
High Resolution Cell Cycle and Apoptosis Analysis in a Two Color Fluorescence Plot O. Herault and C. Vignon. Univ. Hosp. of Tours, CNRS UMR 7292 GICC, Tours.
Oral Session Abstracts
Cell Sorting and Selection B5
126
Poster Session Abstracts
Optimization of Flow Cytometric Detection and Sorting of Transgenic Plasmodium Parasites by Selection of Optical Filters I. Vorobjev, K. Buchholz, P. Prabhat and N. Barteneva. Moscow State Univ., Harvard Sch. of Publ. Hlth., Semrock Inc., Rochester, NY, Boston Children’s Hosp. and Harvard Med. Sch.
Speaker/Author Index
“B” references the board number.
ISAC 2013 Program and Abstracts
45
Congress Overview
Computation and Informatics
Special Lectures
Diagnostics
Saturday, 18 May
Facility Management
Sunday, 19 May
Flow Cytometry Instrumentation
B6
B7
Wednesday, 22 May
Tuesday, 21 May
Monday, 20 May
B8
127
128
129
An Event-Level Relational Database for Flow Cytometry Data J. Cavenaugh, A. Straw, T. Pawlicki, J. Rebhahn and T. Mosmann. Univ. of Rochester.
Multidimensional Data Visualization Tools for Highly Multiparametric Analysis of Signaling Pathways by Mass Cytometry J. De. Deepath Med., Palo Alto, CA.
Chromocyte: An Online Resource for the Flow Cytometry Community A.G. Pockley. Nottingham Trent Univ. and Chromocyte Ltd., Sheffield, U.K.
B9
130
Microfabricated Square Channels for Two Dimensional Acoustically Focused Flow Cytometry T. Woods and S.W. Graves. Univ. of New Mexico.
B10
131
Using the Flexibility of the BD Influx™ Platform for the Development of a Stream Monitoring and Correction Solution S. Dervish, S. Allen, F. Kao and A. Smith. Centenary Inst., Sydney.
B11
132
Simplifying Multiparametric Analysis Further on Microcapillary Flow K. Gillis, E. Santarelli, A. Barican, P. de Borja, D. Luong, A. Khan, J. Clor, R. Pittaro and R. Lefebvre. EMD Millipore, Hayword, CA.
B12
133
Cytometry and Microscopy Aboard the International Space Station P. Todd, M. Kurk, N.S. Logan, S. Moyers, J. Vellinger, T. Maleki Jafarabadi and J.F. Leary. Techshot Inc., Greenville, IN and Purdue Univ.
Immunology 134
Poster Session
B13
Apoptosis as Modulating Factor of CD8+ T Lymphocytes in Human Cutaneous Leishmaniasis R. Nogueira, C. Cunha, A. Gomes-Silva, A.M. Da-Cruz, A. Schubach, M.I. Pimentel, S. Mendonça, M. Lyra and Á.L. Bertho. Oswaldo Cruz Inst.-Fiocruz and Evandro Chagas Clin. Res. Inst., Rio de Janeiro.
Oral Session Abstracts
Commercial Tutorials & Exhibits
Microelectro-Mechanical Systems (MEMS) and Microfluidics B14
135
Acoustic Manipulation of Liposomes P.P. Austin Suthanthiraraj and S.W. Graves. Univ. of New Mexico.
B15
136
Microfluidic Image Cytometry: Modernizing in Vitro Cell-Based Assays with Microfluidic Technology and Image Cytometry T.H. Yoon and J. Park. Hanyang Univ., South Korea.
Speaker/Author Index
Poster Session Abstracts
Multi-dimensional Image Cytometry
46
B16
137
Development of a Confocal Imaging Based Immunological Synapse Formation Assay to Visualize Bispecific Antibody-Mediated Tumor Cell Killing S. Ludmann, M. Weidner, N. Li, C. Afshari, P. Narayanan and K. Keegan. Amgen, Seattle.
B17
138
Discovering New Ligands with Diverse Signaling Pathways for Old Receptors Y. Wu, P. Tapia, G. Fisher, J.J. Strouse, P. Simons, A. Waggoner, J. Jarvik and L. Sklar. Univ. of New Mexico and Carnegie Mellon Univ.
ISAC 2013 Program and Abstracts
Quantification of Protein Aggregates with Flowsight Imaging Cytometer C. Probst, B. Hall and D. Basiji. Amnis, EMD Millipore, Seattle.
B19
140
Optogenetic Coupling for Calcium Transient Analysis and Cardiotoxicity Testing F. Cerignoli, S. Ray, P. McDonough, M. Mark and J.H. Price. Sanford-Burnham Med. Res. Inst., La Jolla, Vala Sciences Inc., San Diego and UCSD Sch. of Engin.
Special Lectures
139
Congress Overview
B18
Other Biological Applications 141
Automated Imaging of Cell Sorter Aerosol Containment Test Samples: A Dual Bead Method K.J. Acklin, V. Papanna, K.E. Ruisaard, K. Ramirez and K. Clise-Dwyer. Univ. of Texas MD Anderson Cancer Ctr.
Saturday, 18 May
B20
Other Technology Advances 142
Sunday, 19 May
B21
Using Videos to Teach Software M. Aranda, E. Hodges, A. Lewis, M. Stadnisky and A. Treistar. Tree Star Inc., Ashland, OR.
Tissue Cytometry/Morphometry 143
Monday, 20 May
B22
Phenotypic Characterization of Polarized Epithelial Cells in a 3D EpiAirway Culture Model Using Confocal Microscopy S. Ludmann, K. Jen, A. Pirrone, K. Rohrbach, N. Li, C. Afshari, E. Trueblood and P. Narayanan. Amgen, Seattle and Thousand Oaks, CA.
Tuesday, 21 May
Poster Presentations Antigen-Specific Immune Responses CD4+ T Cells Are Source of Antigen Specific IFN-g Production in Whole Blood of Patients with Visceral Leishmaniasis O.P. Singh, R. Kumar, S. Gautam, N. Singh, S. Nylen, D. Sacks and S. Sundar. Banaras Hindu Univ., India, Karolinska Inst. and NIAID, NIH.
B24
145
Simultaneous Assessment of CMV Specificity and Functional Response CD8+ T Cells from Bone Marrow Transplant Recipients O. Maguire, G. Chen, K. O’Loughlin, T. Hahn, P. McCarthy, P.K. Wallace and H. Minderman. Roswell Park Cancer Inst., Buffalo.
Poster Session
144
Wednesday, 22 May
B23
Automated Microscopy Ultra High Throughput Image Cytometry Using Time Delay and Integrate CCD Imaging and Reflective Positioning Autofocus M. Guigli, D. Charlot, B. Azimi, R. Agustin, G.J. Gemmen, A.L. Kellner and J. Price. Sanford-Burnham Med. Res. Inst., La Jolla, UCSD and Vala Sciences Inc., San Diego.
B26
147
Ultra High Throughput Image Cytometry via TDI Scanning G. Gemmen, B. Azimi, R. Agustin, M. Guigli and J. Price. Vala Sciences Inc., San Diego and SanfordBurnham Med. Res. Inst., La Jolla.
Oral Session Abstracts
146
Commercial Tutorials & Exhibits
B25
148
A Quantitative Flow Cytometry Assay Applied to Receptor Occupancy Measurements Helps for Preparation and Pharmacodynamic Monitoring of Clinical Trials for Targeted Drugs P. Poncelet, M-L. and M. Moulard. BioCytex, Marseille and DSAR, Sanofi, Vitry-sur-Seine.
B29
150
Cell Type-Specific Analysis of Oncogenesis D. Galbraith, N. Weng, T. Doetschman, R. Lasken and R. Grindberg. Univ. of Arizona and J. Craig Venter Inst., San Diego.
ISAC 2013 Program and Abstracts
Speaker/Author Index
B27
Poster Session Abstracts
Biomarkers
47
Congress Overview
151
Analysis of Immunohistological Images of Stromal Cell Populations in Ultrasound Guided Biopsies of Synovium to Help Predict Patient Outcomes in Rheumatoid Arthritis D. Hardie, J. Turner, K. Raza, C. Buckley and A. Filer. Univ. of Birmingham, U.K.
Special Lectures
B31
152
A Flow Cytometry-Based Method for Enumeration of Foxp3+ Regulatory T Cells in Blood Samples Collected and Stabilized in Cyto-Chex BCT Suitable for Use in Central Laboratories R. Janani, G. Fesseha and T. Robins. Quest Diagnotics Lab., Valencia, CA.
B32
153
Novel Method for the Identification of Endothelial Microparticles Using ImageBased Flow Cytometry and CDd144 A. Venable, R. Williams and B. McFarlin. Univ. of North Texas, Denton.
B33
154
Global Implementation of Flow Cytometry Instrument Standardization by Covance Central Laboratory Services L. Du, V. Glutz, V. Holl, V. Litwin, M. Edinger, J. Hildmann and J. Batchelder. Covance (Asia) Pte Ltd., Singapore, Covance (Geneva) Inc., Covance (Indianapolis) Inc. and BD Biosciences, San Jose, CA.
B34
155
A Cross-Platform Validation Study of EGFR-Activating Mutations in Non-small Cell Lung Carcinoma Cells S. Bokhari, W-R. Lie and T. Baginski. EMD Millipore Corp./ Merck KGaA, St. Charles, MO.
B35
156
A Flow Cytometric Approach to Monitoring Cell Health and Productivity of a Recombinant Human Cell Line in Bio-Pharmaceutical Development M. Gottlieb, J.C. Chuang and F. Boldog. Shire HGT, Lexington, MA.
B36
157
Implementation of a Flow Cytometric Eosinophil CD11b Expression Assay for Clinical Application R. Wnek , M. Tseng, T. Natasha, D. Gallagher, C. Cilissen, R. Weiner and D. Wu. Merck , Rahway, NJ.
B37
158
A Micromethod for Bead-Based Microarray Immunoassays Enabling Clinical Translational Research Studies C. Baker, S. Secor-Socha, D. Roumanes, T. Mosmann and S. Quataert. Univ. of Rochester.
B38
159
Lightening Up the Cellular-Level Reactive Oxygen Species Using Upconversion Sensitized Ruthenium Biosensors J. Zhao, R. Zhang, L. Zhang, Y. Lu, E.M. Goldys and D. Jin. Macquarie Univ., Australia.
Poster Session
Wednesday, 22 May
Tuesday, 21 May
Monday, 20 May
Sunday, 19 May
Saturday, 18 May
B30
Biopharmaceutical Applications 160
Commercial Tutorials & Exhibits
B39
A High-Throughput Flow Cytometry-Based Method for the Detection of AntigenSpecific T Lymphocyte Activation in Non-human Primates for Pre-clinical Drug Development Assessments M. Bernard, G. Rao, J. Loffredo, A. Suri, H. Haggerty and W. Freebern. Bristol-Myers Squibb, New Brunswick, NJ and Princeton, NJ.
Speaker/Author Index
Poster Session Abstracts
Oral Session Abstracts
Cell Proliferation and Death
48
B40
161
Differential Homing and Senescene of CD8+ T-Lymphocytes from Elderly Humans O. Onyema, I. Bautmans, M. De Waele, J. Aerts and T. Mets. Vrije Univ. and Univ. Ziekenhuis, Brussels.
B41
162
Induced Germination of B. atrophaeus Spores by Singlet Oxygen-Sensitizing Cationic Oligo-Phenylene Ethynylenes H. Pappas and D. Whitten. Univ. of New Mexico.
B42
163
Automated Quantification of Budding Saccharomyces cerevisiae Using an Image Cytometry Method L. Chan, D. Laverty, A. Kury, D. Kuksin, A. Pirani and K. Flanagan. Nexcelom Bioscience LLC, Lawrence, MA.
ISAC 2013 Program and Abstracts
B45
166
Bradykinin Functions in Bone Marrow Metatastis H. Ulrich, C. Lameu and M. Ratajczak. Univ. of São Paulo and Univ. of Louisville.
B46
167
Detection and Evaluation of Cellular Phenomena in the Breast Cancer Cell Line Resistance to Cisplatin J. Markovic, B. Krunic, N. Apostolova, S. Bañuls and F.V. Pallardo. Univ. of Valencia, Spain.
B47
168
Improved Click Chemistry Demonstrating EdU Cell Proliferation with GFP Expressing Cells and R-PE Based Immunophenotyping C. DeMarco, J.A. Bradford, S. Clarke, R. Deveny, K. Gee, S. Grecian and U. Singh. Life Technologies, Eugene, OR.
B48
169
Investigating Mitochondrial Changes during Autophagy and Apoptosis Using Microcapillary Cytometry K. Gillis, J. Clor and K. Tyagarajan. EMD Millipore, Hayward, CA.
B49
170
Measure of Cell Cycle with the Tali Image Instrument Using a Broad Range of Dyes T. Lopes, L. Roh and M. Garcia. Fed. Inst. of Technol., Lausanne.
Tuesday, 21 May
Characterization of C22:0 and Saturated Very Long Chain Fatty Acids (C24:0; C26:0)-Induced Cell Death by Microscopical and Flow Cytometric Methods on Human Neuronal Cells (SK-N-BE) A. Zarrouk, H. Iddir, M. Yousfi, T. Nury, C. Gondcaille, M. Hammami and G. Lizard. Univ. of Monastir Fac. of Med., Tunisia and Univ. of Bourgogne, INSERM, Fac. of Sci. Gabriel, Dijon, France.
Monday, 20 May
165
Sunday, 19 May
B44
Saturday, 18 May
Probing the Impact of Bystander Cells on Irradiated Cells in Mixed Co-cultures B. Gerashchenko and R. Howell. R.E. Kavetsky Inst. of Exptl. Pathol., Oncol. and Radiobiol., Kyiv, Ukraine and New Jersey Med. Sch. Cancer Ctr., Newark.
Special Lectures
164
Congress Overview
B43
Cell Sorting and Selection
B52
173
Comparison of Proliferation Markers CD71, Ki-67, and Pyronin Y in Combination with Hoechst 33342 N. Hanson, J. Brown, P. Lopez and M. Schober. New York Univ. Langone Med. Ctr.
B53
174
High Throughput Chip-Based Cellular Sorting via Acoustically Enhanced Magnetophoresis L. Gao, C.W. Shields IV, D.M. Murdoch, B.B. Yellen and G.P. Lopez. Duke Univ., Res. Triangle Material Res. Sci. and Engin. Ctr. and Sch. of Med., Duke Univ.
B54
175
Microvesicle Detection and Cell Sorting V. Toxavidis, J. Tigges and K. Groglio. Beth Israel Deaconess Med. Ctr.
B55
176
Use of Near-IR Detection in Flow Cytometry M. Bigos, D. Parks and T. Schmidt. Stanford Univ.
B56
177
Assessing Sample Behavior in the Context of Sorter Performance Using a Dispersion Index J. Trotter and S. Iyer. BD Biosciences, San Jose, CA.
B57
178
Performance Study of the Closed Piezo-Based Cell Sorter (CyFlow® Sorter) J. Klose, D. Köhler and V. Ost. Partec GmbH, Münster.
ISAC 2013 Program and Abstracts
Speaker/Author Index
Single Cell Sorting for Cancer Stem Cells Enrichment in Hepatocellular Carcinoma E. Trombetta, F. Colombo, S. Mazzucchelli, A. Cattaneo, D. Prati, P. Rebulla and L. Porretti. IRCCS Fndn. Ca’ Granda Policlin., Milan and Hosp “”A. Manzoni”” Lecco, Italy.
Poster Session Abstracts
172
Oral Session Abstracts
B51
Commercial Tutorials & Exhibits
Advances in Fluorescence Decay Analysis: New Techniques for Use in Cytometers of All Shapes and Sizes R. Cao, M.A. Naivar and J. Houston. New Mexico State Univ. and DarklingX, Los Alamos.
Poster Session
171
Wednesday, 22 May
B50
49
Congress Overview
179
Study of Platelet-Derived Microparticles Using the BD FACSVerse™ Flow Cytometer L. Yu and Y. Wang. BD Biosciences, San Jose, CA.
B59
180
Importance of Quality Control to Follow-Up and Compare Instrument Performance for Microparticle Analysis by Flow Cytometry: Correlation to Microparticle Count N. Bailly, P. Poncelet, B. Devalet, S. Robert, R. Lacroix, J-M. Dogné, F. DignatGeorges, B. Chatelain and F. Mullier. CHU Mont-Godinne, Yvoir, Belgium, BioCytex, Marseille, Univ. de la Méditerranée, France and Univ. of Namur, Belgium.
B60
181
Accurate Detection of Counting Beads for Cell-Derived Microparticle Enumeration by Flow Cytometry N. Fisher, M. Mooberry, S. Javardi, R. Zucker and N. Key. Univ. of North Carolina at Chapel Hill, Stratedigm Inc., San Jose, CA and U.S. EPA, Research Triangle Park, NC.
Sunday, 19 May
B61
182
Identification and Analysis of Circulating Endothelial Microparticles as a Potential Biomarker of Drug-Induced Vascular Injury S. Sokolowski, A. Shen, L. Obert, T. Wisialowski, M. Lawton, P. Nugent, T. Swanson, S. Portugal, C. Rief and B. Enerson. Pfizer Inc., Groton, CT.
B62
183
Flow and Imaging Cytometry Characterization of Microparticles: Analysis and Sorting on the Basis of Size and Fluorescence N. Barteneva, E. Fasler-Kan, L. Duckett and I. Vorobjev. Boston Children’s Hosp., Univ. of Basel, BD Biosciences Inc., San Jose, CA and Moscow State Univ.
Tuesday, 21 May
Saturday, 18 May
Special Lectures
B58
Monday, 20 May
Cell-Derived Microvesicles
Poster Session
Wednesday, 22 May
Clinical Trials B63
184
The Applications of Acridine Orange in Cytometry F. Samani, P. Khosravani, E. Jan Zamin and M. Ebrahimi. Royan Inst. for Stem Cell Biol. and Technol., Tehran.
B64
185
Simultaneous Restorations of Foxp3+ Treg, Type 1-Like Treg and B Cells by Anti-TNF Therapy for IBD Z. Li, S. Vermeire, D. Bullens, M. Ferrante, K. Van Steen, M. Noman, P. Rutgeerts, J. Ceuppens and G. Van Assche. Catholic Univ. of Leuven and Univ. of Liege, Belgium.
B65
186
An Approach to Qualifying Flow Cytometry Panels J. Hill, E. Dearstyne, T. Beckett, N. Purdue and S. Apone. Dendreon, Seattle.
Speaker/Author Index
Poster Session Abstracts
Oral Session Abstracts
Commercial Tutorials & Exhibits
Computation and Informatics
50
B66
187
Creating Standards for InstrumentXML and PanelXML N. Ostrout, J. Katz, M. Stadnisky, J. Quinn and A. Treister. Fluorish LLC, FlowJo LLC and Tree Star Inc., Ashland, OR.
B67
188
Variance-Stabilized Meta-Clustering in Flow Cytometry A. Azad, B. Rajwa and A. Pothen. Purdue Univ.
B68
189
Gating the Gate Makers: How to Decide Whose Gates Are Bright, and Whose Are Dim in a High-Throughput Manner J. Quinn, J. Almarode, M. Stadnisky, I. Taylor and A. Treister. Tree Star Inc. and FlowJo LLC, Ashland, OR.
B69
190
Generative Modeling of F-Actin in Cells to Understand Drug-Induced Cytoskeletal Changes J. Kinser, T. Turbyville, K. Reilly, J. Beutler and S.J. Lockett. Sch. of Phys. and Comput. Sci., George Mason Univ. and Frederick Natl. Lab., MD.
B70
191
A Shared Standard for Cytometry and Pathology R. Leif and S. Leif. Newport Instruments, San Diego.
ISAC 2013 Program and Abstracts
Automated Flow Cytometry Data Analysis K. Feher and T. Kaiser. Univ. of Potsdam and DRFZ, Berlin.
B72
193
Automated Flow Cytometry Data Analysis with the OpenCyto Framework J. Ramey, G. Finak, M. Jiang, J. Taghiyar, S. DeRosa, R. Brinkman and R. Gottardo. Fred Hutchinson Cancer Res. Ctr., Seattle and British Columbia Cancer Agcy., Vancouver.
Special Lectures
192
Congress Overview
B71
Cytometry in Resource Poor Settings 194
Reliable and Accurate CD4 T Cell Count and CD4 Percent of the New Portable Flow Cytometer Cyflow Minipoc M. Nasi, S. De Biasi, E. Bianchini, L. Gibellini, M. Pinti, T. Scacchetti, T. Trenti, V. Borghi, C. Mussini and A. Cossarizza. Univ. of Modena and Reggio Emilia, NOCSAE Baggiovara, Modena and Azienda Ospedaliero-Univ. Polyclin. of Modena, Italy.
Saturday, 18 May
B73
Sunday, 19 May
Diagnostics
Determining Reference Bead Concentration and Fluorescence Intensity for Quantitative Flow Cytometry at 660 nm and 760 nm P. DeRose, A. Gaigalas and L. Wang. NIST, Gaithersburg, MD.
B77
198
Characterization of Two Human CD4+ Lymphocyte Preparations for Quantitative Flow Cytometry L. Wang, M. Wang, H-J. He, M. Misakian, I. Turko and K. Cole. NIST, Gaithersburg, MD.
B78
199
Assessment of Myeloid Nuclear Differentiation Antigen in Myelodysplastic Syndrome and Acute Myeloid Leukemia K.T. Soh and P.K. Wallace. Univ. at Buffalo, SUNY and Roswell Park Cancer Inst., Buffalo.
B79
200
Six Color-Single Tube Analysis of Minimal Residual Disease in Paediatric B Lineage Acute Lymphoblastic Leukemia on Paired Mid-induction Peripheral Blood and Bone Marrow Samples: Can Peripheral Blood Replace Bone Marrow Aspirate Sample? M.U. Sachdeva, K. Bommannan, P. Bose, N. Varma, D. Bansal and R.K. Marwaha. Postgrad. Inst. of Med. Educ. & Res., Chandigarh, India.
B80
201
Salivary Cytomics — A Useful Clinical Diagnostic Tool for Dental Professionals Z. Chen and F.Y.S. Hou. Peking Univ. Sch. of Stomatol. and Marquette Univ., Milwaukee.
B81
202
Transfix®/EDTA Stabilization of Leukocytes in Cerebrospinal Fluid for Flow Cytometric Screening of Patients with Suspected Leukemic Conditions T. Almond, D. Harrison, M. Crawford and U. Johansson. Caltag Medsystems Ltd., Buckingham and Bristol Royal Infirm., U.K.
B82
203
Development of 8 Color Panel for Lymphoma Diagnostics with Minimal Compensation Requirements I. Vorobjev and O. Khoudoleeva. Moscow State Univ. and GeneTechnol., Moscow.
ISAC 2013 Program and Abstracts
Speaker/Author Index
197
Poster Session Abstracts
B76
Oral Session Abstracts
Flow Cytometry Detection of LAMP2 Protein in Danon Disease — A Rare X-Linked Cardiomyopathy O. Pelak, J. Sikora, L. Krol, F. Majer, L. Dvorakova, H. Vlaskova, T. Honzik, T. Palece, M. Kubanek and T. Kalina. Charles Univ. in Prague and Gen. Univ. Hosp. and St. Anne’s Univ. Hosp. Brno, Czech Republic.
Commercial Tutorials & Exhibits
196
Poster Session
B75
Wednesday, 22 May
Investigation of Protein-Coated Particle Aggregation Using Scanning Flow Cytometry A. Polshchitsin, V. Nekrasov, A. Chernyshev and V. Maltsev. Inst. of Chem. Kinet. and Combustion, SB RAS, Novosibirsk, Russia.
Tuesday, 21 May
195
Monday, 20 May
B74
51
Congress Overview Special Lectures
B83
204
Analysis of Skeletal Muscle: Correlated Quantification of Mitochondrial Metabolic Enzymatic Activities and Fiber Type-Specific Biomarkers P. McDonough, D. Reiner, T. Kostrominova and R. Haas. Vala Sciences Inc., San Diego, WM&G Consulting, Imperial Beach, CA, Sch. of Med., Northwest, Univ. of Indiana and UCSD.
205
Saturday, 18 May
Extended NK Cells Phenotyping in Patients with Acute Myeloid Leukemia G. Bouvier, F. Orlanducci, C. Fauriat, E. Gautherot, F. Montero Julian, C. Arnoulet and D. Olive. Beckman Coulter Life Sci. and Inst. Paoli Calmettes, Marseille.
B85
206
Correlation of Oxidant Status and Molecular Damage with Disease Activity Score in Patients with Rheumatoid Arthritis S. Kundu, S. Datta, P. Ghosh, A. Ghosh, S. Chattopadhyay and M. Chatterjee. Inst. of Post Grad. Med. Educ. & Res., Kolkata and Bhabha Atomic Res. Ctr., Mumbai.
B86
207
Significant Antigens for Detection of Minimal Residual Disease in Patients with Mantle Cell Lymphoma Using Flow Cytometry Approach J. Chovancova, M. Doubek and J. Mayer. Masaryk Univ. Brno, Central European Inst. of Technol. and Fac. Hosp. Brno, Czech Republic.
B87
208
Detection of Elevated Monocyte Proinflammatory Cytokine Responses by Flow Cytometry in Previously Cryopreserved Samples from HIV+ Individuals at Risk for Cardiovascular Disease E. Jalbert, N. Parikh, T. Seto, D. Chow, L. Ndhlovu, C. Shikuma and J. Barbour. Hawaii Ctr. for HIV/AIDS, Honolulu and The Queen’s Med. Ctr., Univ. of Hawaii.
Tuesday, 21 May
Monday, 20 May
B84
Sunday, 19 May
Disease Progression Monitoring
DNA Damage and Repair
Poster Session
Wednesday, 22 May
B88
209
Quantitative Imaging Analysis of Replication - Vis-a-Vis DNA Damage-Sites in Cells Exposed to DNA Targeting Anticancer Drugs and Oxidative Stress J. Dobrucki, K. Berniak, P. Rybak, T. Bernas, A. Waligórska, M. Zarebski, E. Biela, H. Zhao and Z. Darzynkiewicz. Jagiellonian Univ., Poland, Nencki Inst. of Exptl. Biol., Polish Acad. of Sci., Warsaw and New York Med. Col.
B89
210
Establishment of Flow Cytometry Shared Resource at Children’s Research Institute N. Loof. Children’s Res. Inst. at Univ. of Texas Southwestern.
Commercial Tutorials & Exhibits
B90
211
Algorithm for Calculating the Utilization and Efficiency of Sorting Service A. Petrunkina. Univ. of Cambridge and Univ. of Vet. Med. Hannover.
B91
212
Identifying Challenges, Opportunities, and Strategies for Core Operations T. Fallows and H. Lorenz. iLab Solutions, Boston.
Oral Session Abstracts
Facility Management
B92
213
The Purdue Cytometry Email Discussion List J.P. Robinson and B. Rajwa. Purdue Univ.
Poster Session Abstracts
Flow Cytometry Instrumentation 214
Detection of the Fluorescence Lifetime of Green Fluorescent Protein Expressed in Yeast Cells with a Time-Resolved Flow Cytometry F. Crawford, B. Sands, P. Jenkins, R. Cao, W. Peria, R. Brent and J. Houston. New Mexico State Univ. and Fred Hutchinson Cancer Res. Ctr., Seattle.
Speaker/Author Index
B93
52
ISAC 2013 Program and Abstracts
218
The Importance of Area Scaling with FACS DIVA Software D. Haviland and A. Hazen. Methodist Hosp. Res. Inst., Univ. of Texas Hlth. Sci. Ctr. at Houston.
B98
219
Nano-View Version II: An Improved Novel Approach to Microparticle Cell Sorting V. Toxavidis, J. Tigges and K. Groglio. Beth Israel Deaconess Med. Ctr.
B99
220
8 Way Fluorescence-Activated Cell Sorting on the BD Influx™ S. Dervish, F. Kao, S. Allen and A. Smith. Centenary Inst., Sydney.
B100
221
A High Throughput Flow Cytometric Assay for Rapid Quantitation and Detection of Mouse IgG R. Danielzadeh and M. Krusemeier. Charisela Technol. Inc., Menlo Park, CA and UCSF.
B101
222
Detection of Brother of the Regulator of Imprinted Sites Expression in Peripheral Blood Neutrophils by Flowcytometry in Benign and Malignant Breast Lesions N. El Sharkawy, W.M. Radwan, E.S. Eissa, S.H. Kandil and A.M. Kamel. NCI, Cairo Univ. and Fac. of Med., Menofeya Univ., Egypt.
B102
223
Enhanced Far-Red Fluorescence Sensitivity in CCD Camera-Based Cytometers Increases Threshold Antibody Titration B. McFarlin, K. Clise-Dwyer, K.E. Ruisaard, K.J. Acklin and A. Venable. Univ. of North Texas, Denton and Univ. of Texas, Houston.
B103
224
Withdrawn.
B104
225
Sorted or Agonized? H. Ulrich, I. Andrae, L. Henkel, D. Busch and M. Schiemann. Tech Univ. Munich.
B105
226
Concentration Measurements Using Acoustic Cytometry J. Bradford, B. Dubbels, A. Anderson and Y-Z. Zhang. Life Technologies, Eugene, OR.
B106
227
Low Cost Precision Pulsed LED for Flow Cytometer Calibration in Statistical Photoelectron Units J. Wood. Wake Forest Univ.
B107
228
Improved Light Scatter Detection for Small Particle Analysis and Counting D. Houck, B. Cao and D. Lindseth. BD Biosciences, San Jose, CA.
B108
229
Optimal Baseline PMT Voltages for Multicolor Staining: Optimizing CST Settings for Cellular Samples B. McLaughlin. Univ. of California, Davis.
Oral Session Abstracts
B97
Commercial Tutorials & Exhibits
Acoustically Enhanced Flow Cytometry for Remote Plankton Monitoring D.M. Kalb, R.J. Olson, H.M. Sosik, M.E. Piyeasena and S.W. Graves. Univ. of New Mexico and Woods Hole Oceanographic Instn.
Poster Session
217
Wednesday, 22 May
B96
Tuesday, 21 May
Utilizing Plasmon Surface Resonance for Flow Cytometry M. Buescher, A. Esslinger, J. Krieg, C. Peth and M. Nagel. Miltenyi Biotec, Bergisch Gladback, Germany.
Monday, 20 May
216
Sunday, 19 May
B95
Saturday, 18 May
Polarizing Light-Scattering Profile – Advanced Characterization of Non-spherical Particles with the Scanning Flow Cytometry D. Strokotov, I. Polshchitcina, A. Moskalensky, V. Nekrasov, A. Chernyshev and V. Maltsev. Inst. of Chem. Kinet. and Combustion, SB RAS and Novosibirsk State Univ., Russia.
Special Lectures
215
Congress Overview
B94
Hematological Disorders 230
Important Role of Flow Cytometry Study in Diagnosis of Angioimmunoblastic T-Cell Lymphoma X. Zhang. Geisinger Hlth. Syst., Wilkes-Barre, PA.
B110
231
Withdrawn.
Poster Session Abstracts
B109
Speaker/Author Index
ISAC 2013 Program and Abstracts
53
Special Lectures
Novel Approach Using Imaging Cytometry to Monitor Red Blood Cell Surface Area Loss and Splenic Retention M. Nguyen, I. Safeukui, P.A. Buffet, G. Deplaine, S. Perrot, V. Brousse, A. Ndnour, O. Mercereau-Puijalon, P.H. David, G. Milon and N. Mohandas. Inst. Pasteur, INSERM/UPMC, AP-HP Pitie-Salpetriere Hosp., AP-HP, Necker Hosp., Paris and New York Blood Ctr.
B112
233
Saturday, 18 May
Congress Overview
232
Combined Cell- and DNA-Based Analysis of Genetic Abnormalities in Multiple Myeloma D. Alpar, B. Kajtar, D. de Jong, S. Savola, P. Jakso, L. Kereskai, L. Pajor and K. Szuhai. Univ. of Pecs, Hungary, Leiden Univ., Netherlands and MRC-Holland, Amsterdam.
B113
234
Plasma Cell Heterogeneity in Normal Bone Marrow J. van Velzen, M. Minnema and A. Bloem. Univ. Med. Ctr. Utrecht, Netherlands.
B114
235
Separating Platelets and RBCs by FSC Amplitudes M. Krockenberger. Abbott Hematol., Santa Clara, CA.
Sunday, 19 May
B111
Wednesday, 22 May
Tuesday, 21 May
Monday, 20 May
High Content Analysis B115
236
Application of 5 Multivariate Classification Methods to Compare Conventionally Analyzed Multidimensional Flow Cytometry Data A. Donnenberg, V.S. Donnenberg and D. Normolle. Univ of Pittsburgh Sch. of Med. and Grad. Sch. of Publ. Hlth.
B116
237
Modeling Subcellular Distribution from Chemical Structure D. Sullivan, E. Cesanek, G.R. Rosania and R.F. Murphy. Carnegie Mellon Univ., Vassar Col. and Univ of Michigan.
B117
238
Manipulating 30-50 Parameters in Real Time: High Content Analysis of Flow Cytometry Data J.P. Robinson. Purdue Univ.
B118
239
A Novel High Content Microscopic Method for Mitochondrial Morphometry A. Leonard, C. Beeson and B. Rohrer. Med. Univ. of South Carolina.
B119
240
CyTOFAnalyzer: A High Content Analysis Pipeline Developed via the PlateAnalyzer Environment J.P. Robinson, V. Patsekin and B. Rajwa. Purdue Univ.
Oral Session Abstracts
Commercial Tutorials & Exhibits
Poster Session
High Throughput Instrumentation B120
241
Mega-Multiplexing Suspension Arrays J. Lu, Y. Lu, J. Zhao, E.M. Goldys, R.C. Leif, J.A. Piper, J.P. Robinson and D. Jin. Macquarie Univ., Australia, Newport Instruments, San Diego and Purdue Univ.
B121
242
Development of FAP Technology: Fluorogen Activated Protein in High Throughput Cytometry D. Gebhard, S. Hallowell, E. Benvenuti and R. Doyonnas. Pfizer Inc., Groton, CT.
B122
243
A Mix-and-Read Cell-Based Assay for Antibody Screening against Epithelial Growth Factor Receptor D. Caracino, W.P. Bowen, D. Onley, P. Wylie and T. Cope. TTP Labtech, Cambridge, MA and Melbourn, U.K.
Speaker/Author Index
Poster Session Abstracts
High Throughput Screening
54
B123
244
Development of a Multiplex Oligonucleotide Ligation-PCRr Assay for the Detection of Shiga Toxin Producing E. coli in the Beef Chain T. Woods, S.W. Graves and A. Deshpande. Univ. of New Mexico and Los Alamos Natl. Lab.
B124
245
Single-Cell Nanotoxicity of Nanobarcoded Superparamagnetic Iron Oxide Nanoparticles Quantified by High-Throughput Scanning Image Cytometry J. Leary and T. Eustaquio. Purdue Univ. and Natl. Ctr. for Toxicol. Res., FDA, Jefferson, AR.
ISAC 2013 Program and Abstracts
247
Development of a Multiplexing, High-Throughput Technique for Initial Cell Screening J. Tasset, P. Hexley, C. Robinson, A. Osterburg, C. Fu and G. Babcock. Univ. of Cincinnati, Shriners Hosps. for Children, Cincinnati and Wright Patterson Air Force Base, Dayton, OH.
B127
248
Differences in Whole Blood Light Scattering from Multiple Laser Excitation Sources: A Rapid No-Lyse, No-Wash Method Using the Attune® Acoustic Focusing Cytometer with Red or Violet Laser Option A. Dickson and J. Bradford. Life Technologies, Eugene, OR.
B128
249
Complex Cell Based Assays with a Novel Imaging Cytometry System O. Sirenko, S. Boege, J. Hesley, D. Henderson, A. Cohen, M. Starodynov, E. Cromwell and P. Comita. Molecular Devices, Sunnyvale, CA.
Sunday, 19 May
B126
Saturday, 18 May
Flow Cytometry as a Drug Screening Platform R. Jepras, P. Shah, M. Patel, E. Sutton and S. Ludbrook. Glaxosmithkline, Stevenage, U.K.
Special Lectures
246
Congress Overview
B125
Image Processing and Analysis Detecton of Internalized Exosomes by Tumor Cells Using Amnis ImageStreamX P. Simms, C. Franzen, V. Volgina and G. Gupta. Loyola Univ. Chicago, Maywood.
B130
251
Cellometer Image Cytometry as a Complementary Analysis Tool to Flow Cytometry for Visual Verification of Gated Cell Populations L. Chan, D. Kuksin, C. Kuksin and J. Qiu. Nexcelom Bioscience LLC, Lawrence, MA and Univ. of Massachusetts Amherst.
B131
252
Effects of Flow Cytometry on the Physical Appearance of Flow Cytometry Professionals C.K. Becker, M. Becker, P.R. Becker and B. Becker. Phoenix Flow Systs. Inc., San Diego.
Tuesday, 21 May
250
Monday, 20 May
B129
Wednesday, 22 May
Immune Monitoring
Functional Defects in the Immune Cells Responses in Erdheim-Chester Disease W. Tsai, K. Davis, J. Estrada-Veras, R. Wang, B. Gochuico, W. Gahl and M. Gadina. NIAMS and NHGRI, NIH.
B134
255
Tumor Exome Analysis Reveals Neo-antigen-Specific T Cell Reactivity in an Ipilimumab-Responsive Melanoma P. Kvistborg, N. van Rooij, M. van Buuren, D. Philips, A. Velds, M. Toebes, L. van Dijk, S. Behjati, H. Hilkmann, D. el Atmioui, M. Nieuwland, M. Stratton, R. Kerkhoven, C. Kesmir, J. Haanen and T. Schumacher. Netherlands Cancer Inst., Amsterdam, Wellcome Trust Sander Inst., Cambridge and Utrecht Univ., Netherlands.
B135
256
Altered Mitochondrial Functional Response to Activation in T Cells of the Neonate G. Meszaros, A. Kaposi, B. Gyarmati and B. Vasarhelyi. Semmelweis Univ., Hungarian Acad. of Sci. and Uzsoki Street Hosp., Budapest.
B136
257
Assays to Measure Natural Killer Cell Function in Cynomolgus Macaques C. Donovan, S. Haskett, C. Homiski and C. Kamperschroer. Pfizer Drug Safety R&D, Groton, CT.
B137
258
Rapid and Semi-automated Monitoring of Antigen-Specific T Cell Immunity M. Herber, M. Niemöller, K. Lange, S. Weber-Lohmann, S. Höher-Peters, M. Assenmacher and A. Richter. Miltenyi Biotec GmbH, Bergisch-Gladbach.
ISAC 2013 Program and Abstracts
Speaker/Author Index
254
Poster Session Abstracts
B133
Oral Session Abstracts
Investigation of Immune System Involvement in Prosthetic Implant Failure with Polychromatic Flow Cytometry P. Court, D. Ebreo, S. Donell and S. Carding. Inst. of Food Res., Norwich and Norfolk and Norwich Univ. Hosp., U.K.
Commercial Tutorials & Exhibits
253
Poster Session
B132
55
Congress Overview Special Lectures Saturday, 18 May Sunday, 19 May
B138
259
Using Image-Based Flow Cytometry to Simultaneously Measure Granulocyte Phagocytosis and Oxidative Burst: A Beginner’s Guide R. Williams, A. Venable and B. McFarlin. Univ. of North Texas, Denton.
B139
260
Following Reconstitution of CMV Immunity in Allogeneic Hematopoietic Cell Transplantation Patients K. Jacobsen, L. Brix, D. Pan, C. Halgreen, T. Hahn, P. McCarthy and P.K. Wallace. Immudex, Copenhagenk and Roswell Park Cancer Inst., Buffalo.
B140
261
Mass Cytometry for Multiparametric Immunophenotyping of Seasonal Flu Vaccine Recipients M. Leipold, M. Davis and H. Maecker. Stanford Univ.
B141
262
Multi-Color Flow Analysis of Subsets of PBMC for TLR Ligand Stimulation S. Chitta, H-K. Lee, P. Quintel, G. Singh, J. Rosernberg and S. Singh. Imgenex Corp., San Diego.
B142
263
The Prevalence of Intracellular Galectin-1-Expressing Lymphocytes in Umbilical Cord Blood in Comparison with Adult Peripheral Blood G. Toldi, S. Kollár, J. Rigó, Jr., T. Tulassay and B. Vásárhelyi. Semmelweis Univ., Hungary.
B143
264
Simultaneously Measuring 18 Human Cytokines on a Conventional Flow Cytometer Y. Song and S. Luo. YSL Bioprocess Develop. Co., Pasadena.
B144
265
Technical Advancements Allowing Improved Selection of True, Viable, Regulatory T Cells J. Tigges, V. Toxavidis and K. Groglio. Beth Israel Deaconess Med. Ctr.
B145
266
Identification and Functional Characterization of Four Subsets of Renal Mononuclear Phagocytes in Healthy and Diseased Kidney X. Wang, Q. Cao, Y. Wang and D. Harris. Westmead Millennium Inst., Australia and Univ. of Sydney.
B146
267
Quality Assurance for Bone Marrow Aspirate Specimens from Non-human Primates C. Porretta, R. Siggins II, S. Cormier and G. Bagby. LSU Hlth. Sci. Ctr., New Orleans.
B147
268
18-Color Flow Cytometry: Immunophenotyping Cellular Infiltration during Flavivirus Encephalitis in the Mouse T. Ashhurst, C. van Vreden, M. Karimi Azardaryany, K. Lundsten, S. Allen, S. Dervish, F. Kao, A. Smith and N. King. Univ. of Sydney and BioLegend, San Diego.
B148
269
The Regulatory Role of NLRC5 in MHC I Expression in B Lymphocytes A. Veres, L. Mátyus, S. Benko and A. Jenei. Univ. of Debrecen, Hungary.
B149
270
Modulating in Vivo T-Cell Activation: 15 Color Immunophenotyping, Cytokine Analysis, and Cellular Redistribution K. Lundsten, M. Tam, J. Ampudia, N. Urbina, J. Ransom and G. Lay. BioLegend, San Diego.
B150
271
A Longitudinal Flow Cytometry–Based Study on Phenotypic Changes of Cryopreserved Murine T Cells Using the BD FACSVerse™ System Y. Wang, S. Cai, W. Feng and R. Jana. BD Biosciences, San Jose, CA and Stanford Univ. Sch. of Med.
B151
272
Defining CD4+ T-Cell Subsets Using Probability State Modeling M. Inokuma, J. Trotter, E. Hill, B. Hunsberger, M. Munson, D. Herbert, C. Bray, S. Ghanekar, V. Maino and C.B. Bagwell. BD Biosciences, San Jose, CA and Verity Software House, Topsham, ME.
B152
273
Immunophenotyping in Clinical Translational Studies Using Brilliant Violet Dye Conjugates D. Roumanes, C. Baker, S. Secor-Socha, Y. Qi, A. Dunham, T. Mosmann and S. Quataert. Univ. of Rochester Med. Ctr.
Speaker/Author Index
Poster Session Abstracts
Oral Session Abstracts
Commercial Tutorials & Exhibits
Poster Session
Wednesday, 22 May
Tuesday, 21 May
Monday, 20 May
Immunology
56
ISAC 2013 Program and Abstracts
Congress Overview
Infectious Diseases B153
274
Special Lectures
A Flow Cytometric Method for Intracellular Detection of HIV-1 P24 Core Antigen Used to Investigate the Prevention of HIV-1 Transfer from Dendritic Cells to CD4 T Cells by Neutralizing Antibodies V. Holl, B. Su, A. Lederle, M. Peressin, V. Glutz, M. Lambotin and C. Moog. Covance, Geneva and INSERM U748, Strasbourg.
Live Cell Imaging/Tracking
B155
276
Determining RNA Expression at the Single Cell Level in Live Cells Using Flow Cytometry D. Weldon, A. Ko, Y. Williams, L. White, L. Armstrong and V. Koong. EMD Millipore, Temecula, CA.
B156
277
Simultaneous Tracking of Cell Type, Viability, Proliferation and Expression of Intracellular Proteins in Co-cultures of Leukemia and HS-5 Stroma Cells Using Multiparameter Flow Cytometry K. Piwocka, P. Podszywalow-Bartnicka, M. Brewinska-Olchowik, L. Bugajski and M. Kusio-Kobialka. Nencki Inst. of Exptl. Biol., Warsaw.
Monday, 20 May
Multiplexing Analysis of Cell Proliferation and Cellular Functions Using a New Multicolor Panel of Fluorescent Cell Proliferation Dyes Z. Diwu, Q. Zhao, Y. Wu and J. Liao. AAT Bioquest Inc., Sunnyvale, CA.
Sunday, 19 May
275
Saturday, 18 May
B154
Microbiology and Aquatic Sciences Scanning Flow Cytometry for Static and Dynamic Characterization of E. coli Cells A. Konokhova, M. Yurkin and V. Maltsev. Inst. of Chem. Kinet. and Combustion, SB RAS and Novosibirsk State Univ., Russia.
B158
279
Moflo Astrios™ Forward Scatter: Cell Sorting of Nano and Large Phytoplankton Simultaneously with High Purity C. Ross, A. Dean and R. Morris. Beckman Coulter Life Sci. Div., Fort Collins, CO.
B159
280
Development of a Plankton Cell Sorter Utilized with the Amnis ImageStream G. Wiegand. Estuary Biophotonics, Baltimore, MD.
Wednesday, 22 May
278
Tuesday, 21 May
B157
Poster Session
Microelectro-Mechanical Systems (MEMS) and Microfluidics
B161
282
Chip Based Impedance Flow Cytometer with Integrated Acoustophoretic Sample Preconditioning C. Grenvall, C. Antfolk, C. Zoffmann Bisgaard and T. Laurell. Lund Univ., Sweden and FOSS Analytical A/S, Denmark.
B162
283
Microfluidic Fluorescence-Activated Cell Sorter Employing ‘Space-Time’ Coding and On-Chip Piezoelectric Actuator. M. Arámbula, S.H. Cho, D. Johnson, R. Alon, P. Buerki, P. Poonka, K. Chuang, Y-H. Lo, Z. Olson and J. Morachis. NanoCellect Biomed. Inc., San Diego, UCSD and Natl. Instruments, Lake Forest, CA.
B163
284
Optofluidic Flow Cytometer Employing Color-Space-Time Coding: On-Chip Multiple Fluorescence Differentiation S.H. Cho, P. Poonka, K. Chuang, P. Buerki, M. Arambula, Z. Olson, J. Hanks, Y-H. Lo and J. Morachis. NanoCellect Biomed. Inc., San Diego, Natl. Instruments Corp., Austin, TX and UCSD.
Poster Session Abstracts
Human Organ-on-a-Chip BioMEMSs Devices for More Rapid Testing of New Multimodal in Vivo Diagnostic Imaging and Therapeutic Strategies J. Leary, P-A. Vidi, C. Cooper and S. Lelievre. Purdue Univ.
Oral Session Abstracts
281
Commercial Tutorials & Exhibits
B160
Speaker/Author Index
ISAC 2013 Program and Abstracts
57
Congress Overview
Multi-dimensional Image Cytometry B164
285
Special Lectures
Rapid Method for Evaluating Micronuclei Formation Using ImageStreamX A.N. White, A. Sullivan, S. Thornton and S. Pfuhler. Cincinnati Children’s Hosp. Med. Ctr. and Procter & Gamble, Mason, OH.
B165
286
High-Throughput Image Analysis Software Applied to High-Content Neuronal Screens D. Logan and A. Carpenter. Broad Inst. of Harvard and MIT.
B166
287
PerFix-nc (No Centrifuge Assay): The New Intracellular No-Wash Assay with Extended Capabilities F. Malergue, L. Khemici and F.A. Montero-Julian. BeckmanCoulter Immunotech, Marseille.
B167
288
Development of Novel Metal-Chelating Polymers for Mass Cytometry D. Majonis, X. Lou, O.I. Ornatsky, V. Baranov and S.D. Tanner. DVS Sciences Inc., Markham and Richmond Hill, ON.
B168
289
A Novel Rapid Apoptosis Assay Based on Thiol Redox Status L. Johansen, S. Kjærulff and M. Skindersø. Chemometec, Allerod, Denmark.
B169
290
New Fluorophore for Violet Laser Excitation J. Bradford, W. Zhou, B. Dubbels, B. Bone, A. Anderson and K. Gee. Life Technologies, Eugene, OR.
B170
291
Brilliant UltraViolet™ Dyes: A New Set of High Sensitivity Fluorescence Reporters for Multicolor Flow Cytometry with a Uv Laser B. Gaylord, Y. Liang, F. Uckert, B. Leonard, H. Li, L. Tran, G. Bartholomew, Y. Chen, F. Luan, S. Widmann and A. Stall. BD Biosciences, San Diego.
B171
292
Monitor Caspase 3/7 Activity without Cell Fixation: A Novel Apoptosis Reagent from Molecular Probes® B. Seredick, J.A. Bradford and M. Olszowy. Life Technologies, Eugene, OR.
B172
293
Reactive Oxygen Probes — A Broad Range of Colors with Easier Labeling and Compatibility with Fixation: Novel CellROXx® Reagents from Molecular Probes® B. Seredick, B. Bone and M. Olszowy. Life Technologies, Eugene, OR.
B173
294
Barcoding with Lipophilic Dyes Can Further Increase the Throughput of Flow Cytometry Screening O. Sharif, J. Gilligan, P. Anderson, C. Trussell, E. Ainscow and J. Joslin. Novartis (GNF), San Diego.
B174
295
Ptychography — Label-Free Imaging of Dividing Cells Using Quantitative Phase Information P. O’Toole, K. Hogg and J. Marrison. Univ. of York, U.K.
Commercial Tutorials & Exhibits
Poster Session
Wednesday, 22 May
Tuesday, 21 May
Monday, 20 May
Sunday, 19 May
Saturday, 18 May
New Probes and Assays
New Software Development
Oral Session Abstracts
B175
296
High-Fidelity Data Transfer: The Secret to Pipelining and Automated Cytometric Analysis M. Stadnisky, J. Quinn, J. Almarode and A. Treister. Tree Star Inc., Ashland, OR.
Speaker/Author Index
Poster Session Abstracts
Other Biological Applications
58
B176
297
The Dipole Potential Influences the Clustering of ErbB Proteins T. Kovacs, A. Szabo, J. Szollosi and P. Nagy. Univ. of Debrecen, Hungary.
B177
298
Flow Cytometric Measurement of the Chromosomal DNA Content and the Genome Size of the Tasmanian Devil B.L. Ng, E. Murchison and D. Adams. The Wellcome Trust Sanger Inst., Cambridge, U.K.
ISAC 2013 Program and Abstracts
B179
300
Using of Combination Cytophotometrical Method for the Determination of Dry Mass, Glycogen and DNA Contents of Hepatocytes in Normal and Cirrhotic Rat Liver A. Chestnova, N. Bezborodkina and B. Kudryavtsev. Inst. of Cytol., St. Petersburg, Russia.
B180
301
Comparison of Viability and Functionality of Sorted Human Dendritic Cells Using Two Types of Flow Cells M. Jaimes, D. Gorgone, D. Ellemore, D. Soni, P. Norton, D. Vrane, S. Waheed, J. Crane and K. Pennebaker. BD Biosciences, San Jose, CA.
Saturday, 18 May
Flow Cytometric Standarization for the Analysis of Microparticles V. Toxavidis, J. Tigges and K. Groglio. Beth Israel Deaconess Med. Ctr.
Special Lectures
299
Congress Overview
B178
Other Clinical Applications
303
The Effect of Nucleotide Supplementation on Atlantic Salmon Intestine J. Bierla, W. Ladno, M. Kamaszewski, T. Ostaszewska, D. Martinez-Puig, C. Chetrit, E. Borda Casas and R. Zabielski. Med. Univ. of Warsaw, Warsaw Univ. of Life Sci. and Bioiberica, Barcelona.
B183
304
Preliminary Results in Tissue Cytometry Investigation of Apelin Influence on Young Rats Digestive System J. Bierla, H. Antushevich, M. Kapica, B. Pawlina, A. Kuwahara and R. Zabielski. Med. Univ. of Warsaw, Polish Acad. of Sci., Univ. of Life Sci. in Lublin, Poland, Univ. of Shizuoka, Japan and Warsaw Univ. of Life Sci.
B184
305
Preliminary Results of Ion Channels Expression in Pyramidal Cells of Prefrontal Cortex 20-Day-Old Rats. J. Bierla, G. Witkowski, W. Ladno, I. Wieczorkowska, B. Greszta and P. Szulczyk. Med. Univ. of Warsaw Fac. of Pharm.
Wednesday, 22 May
B182
Tuesday, 21 May
A Flow Cytometric Study on DEMRON-Mediated Radioprotection in Mice Z. Sinkorova, L. Zarybnicka, L. Navratil and P. Castulik. Univ. of Defence, Czech Tech Univ. and Masaryk Univ. Brno, Czech Republic.
Monday, 20 May
302
Sunday, 19 May
B181
Other Technology Advances
Simultaneous Analysis of Total and Phospho Proteins in Single Cells for Accurate Assessment of Intracellular Cell Signaling Events M. Santos, M. Bader, P. deBorja, A. Barican, D. Luong, R. Lefebvre and M. Hsu. EMD Millipore Corp., Temecula and Hayward, CA.
B187
308
SWOFF — The Unrecognized Sibling of FMO M. Kapinsky. Beckman Coulter Inst., Nittendorf, Germany.
B188
309
Evaluation of Multiplexed RNA Flow Cytometry in HIV-Infected Cells M.B. Hanley, E. Park and V. Maino. BD Biosciences, San Jose, CA.
B189
310
Tumor Histoids: Scalable Production of Living Human Mini-tumors for High Throughput Drug Screening M. Ingram, G. Techy, S.A. Imam, J. Nolan and B. Ward. Huntington Med. Res. Insts., Pasadena, CA and La Jolla Bioengineering Inst.
B190
311
Single Cell Analyses of Natural Killer Cell Development with Fluidigm Microfluidic Devices: A Work in Progress D. Redelman, V. Lombardi, L. Peri and J. Townsend. Univ. of Nevada Sch. of Med., Reno.
ISAC 2013 Program and Abstracts
Speaker/Author Index
307
Poster Session Abstracts
B186
Oral Session Abstracts
The Feasibility of Using Tunable Thin-Film Optical Filters for Excitation - or EmissionScanning Hyperspectral Microscopy P. Favreau, T. Rich, A. Stringfellow, D. Alvarez, P. Prabhat and S. Leavesley. Univ. of South Alabama and Semrock Inc., a Unit of IDEX Corp., Rochester, NY.
Commercial Tutorials & Exhibits
306
Poster Session
B185
59
Congress Overview Special Lectures
Bridging the Gap between Plate Reader Assays and High Content Imaging J. Hesley, S. Boege, B. Wade, J. Dzubay, O. Sirenko, J. Itatani and P. Comita. Molecular Devices LLC, Sunnyvale, CA.
B192
313
EdU Cell Proliferation Improved Compatibility with GFP and Other Fluorescent Proteins S. Clarke, H. Frend, J. Sordet-Dessimoz, U. Singh and K. Gee. Life Technologies, Eugene, OR, Cambridge Univ., U.K. and Fed. Inst. of Technol., Lausanne.
Saturday, 18 May
312
Rare Event Detection B193
314
Progress towards Using Lanthanide Nanoparticles as Isotope Tags for Mass Cytometry P. Cao, A. Halupa, L. Tong, G. Zhao, P. Chattopadhyay, M. Roederer, M. Winnik and M. Nitz. Univ. of Toronto and NIAID, NIH.
Sunday, 19 May
B191
B194
315
RNA Flow Cytometry for the Analysis of Rare Tumor Cells Present in Blood C. Lomas, D. Mittar, W. Zhu, J. Lang and E. Park. BD Biosciences, San Jose, CA and Norris Comprehen. Cancer Ctr., Univ. of Southern California.
316
Linolenic Acid Counteracts TNF Negative Effects on Skeletal Muscle Cells L. Teodori, F. Carotenuto, M.C. Albertini, M. Rocchi, D. Coletti, A. Costa, M. Minieri and Di Niardo. ENEA-Frascati, Rome, Fndn. San Raffaele Pisana, Rome, Univ. of Rome Tor Vergata, Univ. of Urbino “Carlo Bo”, Univ. Pierre et Marie Curie, Paris 6, Univ. of Rome Sapienza and Link Campus Univ. of Rome.
B196
317
Wednesday, 22 May
Functionalization of Decellularized Scaffold for Skeletal Muscle Tissue Engineering A. Costa, P. Aprile, P. Aulino, B. Perniconi, S. Adamo, D. Coletti and L. Teodori. Sapienza Univ. of Rome, Tor Vergata Univ. of Rome, ENEA-Frascati, Rome and Univ. Pierre et Marie Curie, Paris 6.
B197
318
Exploiting Vasopressin Signaling in Muscular Atrophy P. Aprile, A. Costa, B.M. Scicchitano and S. Adamo. Tor Vergata Univ. of Rome, ENEA-Frascati and Sapienza Univ. of Rome.
Signal Transducton
Oral Session Abstracts
Commercial Tutorials & Exhibits
Tuesday, 21 May
B195
Poster Session
Monday, 20 May
Regenerative Medicine
B198
319
PerFix-EXPOSE (Phospho Epitopes Exposure Kit): A New Fast and Easy Procedure for Cell Signaling Analysis F. Malergue, V. Mallet, C. Scifo, A. Van Agthoven and F.A. Montero-Julian. Beckman-Coulter Immunotech, Marseille.
B199
320
Quantitative Microscopic Analysis Reveals Cell Confluence Regulated Divergence of PDGFR-Initiated Signaling Pathways with Logically Streamlined Cellular Outputs A. Szoor, L. Ujlaky-Nagy, J. Szollosi and G. Vereb. Univ. of Debrecen, Hungary.
Small Molecule Discovery
Poster Session Abstracts
B200
321
Development of a Cytomics-Based Mitochondrial Signaling Assay for Hazard Identification of Small Molecule Drug Candidates N. Li, Y. He, H. Ingle, J.P. Robinson, J. Davisson, H. Hamadeh, C. Afshari and P. Narayanan. Amgen, Seattle and Thousand Oaks, CA and Purdue Univ.
Speaker/Author Index
Solid Tumors B201
60
322
Cyclooxygenase-Independent Chemoprevention of Colorectal Cancer by Nonsteroidal Anti-inflammatory Drugs V. Vaish, and S. Sanyal. Panjab Univ., India.
ISAC 2013 Program and Abstracts
324
The Effect of Hypoxia on the Efficiency of Elisidepsin T. Varadi, A. Kiraly, T. Hajdu, J.M. Molina-Guijarro, J. Szollosi, C. Galmarini and P. Nagy. Univ. of Debrecen, Hungary and PharmaMar, Madrid.
B204
325
Integrative Analysis of Genomic and Gene Expression Data of Paired Primary and Metastatic Melanoma M. Balazs, L. Vizkeleti, S. Ecsedi, G. Emri, V. Koroknai, T. Kiss and R. Adany. Med. and Hlth. Sci. Ctr., Univ. of Debrecen, Hungary.
B205
326
Selective Internalization of a Novel Recombinant Human Granzyme B by Membrane Hsp70 Positive Tumor Cells and Its Cytotoxic Consequences: A Novel Therapeutic Approach for Cancer? A.G. Pockley, G. Foulds, M. Gehrmann, B. Doss and G. Multhoff. Nottingham Trent Univ., Univ. of Sheffield, Tech. Univ. Munich and German Res. Ctr. for Envrn. Hlth. Munich.
Sunday, 19 May
B203
Saturday, 18 May
TGFb1 Increased Migration Characteristics and Induced Different Stemness in A549 Cell Fractions Sorted for CD133 Surface Expression and Side Population Phenotype G. Pirozzi, V. Tirino, R. Camerlingo, E. Irollo, R. Montella, F. Paino, G. Sessa, M.V. Carriero, N. Normanno and G. Rocco. INT Fndn. G Pascale and Second Univ. of Naples.
Special Lectures
323
Congress Overview
B202
Monday, 20 May
Standards and Calibration
328
Comparison of LED and Microsphere Methods for Estimating Photoelectron Scales in Flow Cytometers and Use of LED Signals to Compare Efficiencies of PMTs D. Parks, E. Chase, M. Bigos and W.A. Moore. Stanford Univ. and Cytek Develop. Inc., Freemont, CA.
B208
329
Microparticle Analysis by High-Sensitivity Flow Cytometry: Out-of-Routine Detection below the Standardized 0.3µm-eq FSC Cut-Off Is Feasible and Reveals More Small Vesicles P. Poncelet, S. Robert, R. Lacroix and F. Dignat-George. Biocytex, VRCM, INSERM UMR-S1076 and La Conception Hosp., AP-HM, Marseille.
B209
330
Performance Benchmarking the Detection Sensitivity of Image Cytometers Using Fluorescent Glass M. Halter, P. DeRose, G. Cooksey, A.L. Plant and J. Elliott. NIST, Gaithersburg, MD.
B210
331
A Strategy for Comparing Antibody-Based Measurements Using a Cell-Based Reference Standard in Flow and Image-Based Cytometry G. Cooksey, J. Elliott and A. Plant. NIST, Gaithersburg, MD.
B211
332
Characterization of Fluorescence Detection Resolution Limit in Flow Cytometer M. Yan and A. Zhong. BD Biosciences, San Jose, CA.
Commercial Tutorials & Exhibits
B207
Poster Session
A Simple Spreadsheet-Guided Method for Evaluating Cytometer Measurement Linearity and Generating Correction Tables for More Accurate Results D. Parks, M. Bigos and W.A. Moore. Stanford Univ.
Wednesday, 22 May
327
Tuesday, 21 May
B206
Oral Session Abstracts
Stem Cells Multicolour Flow Cytometry Panel for Identification of Human Mesenchymal Stem Cells I.L. Pieper, J.C. Bishop, S. Sultan, C.A. Thornton and G. Morgan. Col. of Med., Swansea Univ., U.K. and Cell Therapy Ltd., Cardiff, U.K.
B213
334
ProtocolNavigator – Interpreting Provenance in Stem Cell Cytometry I. Khan, A. Fraser, M-A. Bray, P. Smith, P. Stephens, A. Sloan, A. Carpenter and R. Errington. Sch. of Med., Cardiff Univ., U.K. and Broad Inst. of MIT and Harvard.
ISAC 2013 Program and Abstracts
Speaker/Author Index
333
Poster Session Abstracts
B212
61
Congress Overview
B214
335
Special Lectures
Vg9Vd2 T Cell Cytotoxicity against Oral Tumors Is Enhanced in the Presence of Monoclonal Antibody B11F12 A. Anand and S. Chiplunkar. Adv. Ctr. for Treatment Res. and Educ. in Cancer, Tata Mem. Ctr., Mumbai.
B215
336
Effect of Silibinin on Stemness Properties in 3D Model of Breast Cancer Cells P. Abdollahi, M. Ebrahimi and N. Motamed. Fac. of Sci., Univ. of Iran and Royan Inst. for Stem Cell Biol. and Technol., Tehran.
B216
337
Phosphorylated CrkL Level in Association to P-Glycoprotein (Pgp) Activity Identifies Imatinib Sensitive Chronic Myeloid Leukemia Patient Samples F.C. Vasconcelos, G.N. Moraes, A.L. Mencalha and R.C. Maia. Brazilian Natl. Cancer Inst., Rio de Janeiro.
Sunday, 19 May
Tissue Cytometry/Morphometry
Monday, 20 May
Tools: Chemical Probes and Fluorescent Proteins
Tuesday, 21 May
Saturday, 18 May
Therapeutics
Toxicology
B217
B218
339
FRET as a Spectroscopic Ruler Revisited with Multiple Fluorophores T. Rente, J. Szollosi, L. Matyus and A. Jenei. Univ. of Debrecen, Hungary.
Detection of Silver and Titanium Dioxide Nanoparticles Using Flow Cytometry Light Scatter and Darkfield Microscopy R. Zucker, L. Degn, J. Ortenzio, and W. Boyes. U.S. EPA, Research Triangle Park.
Wednesday, 22 May
340
Tissue Cytometry Based on Chipcytometry: Strengths, Limitations and Applications C. Hennig and A. Mirenska. Hannover Med. Sch., Germany.
Vaccines
Poster Session
B219
338
Late-Breaking Abstracts
341
The Effect of Alternative Oxygen Environments on Immunotherapeutic Dendritic Cell Vaccines J. Tario, Jr., C. Choi and P. Wallace. Roswell Park Cancer Inst., Buffalo.
B221
343
Plateanalyzer: Gate-Free Comparison of Phenotypes for Automated High Throughput Flow Cytometry J.P. Robinson, V. Patsekin, E. Foo, J. Sturgis and B. Rajwa. Purdue Univ.
B222
344
Plateanalyzer: Tracking Signaling Pathways from Multivariate Flow Cytometry Listmode Data J.P. Robinson, V. Patsekin and J. Sturgis. Purdue Univ.
B223
345
A Single Laser Flow Cytometer: Simplifying Multicolor Immunophenotyping Using Brilliant Violet Dyes J. Rohrer, L. Duckett, B. Gaylord, D.T. Sasaki, A. Stall, A.A. Nguyen and J. Vidal. BD Biosciences Inc.
B224
346
Particle Characterization of Therapeutic Protein Formulations: A Comparison of Flow and Imaging Cytometry with Industry Standard Technologies. K. Kelley, N. Ball, T. Dillon and J. Ferbas. Amgen Inc.
B225
347
Intact Cell-Based Analysis and Screening by Combination of Flow Cytometry and Fluorescence Lifetime Measurement M. Suzuki, K. Koike, I. Sakata, N. Nemoto, T. Sakai and K. Nishigaki. Grad. Sch. of Sci. and Engin., Saitama Univ., Japan.
Speaker/Author Index
Poster Session Abstracts
Oral Session Abstracts
Commercial Tutorials & Exhibits
B220
62
ISAC 2013 Program and Abstracts
B230
352
Orientation of Erythrocytes for Flow Cytometry O. Jakobsson, C. Grenvall and T. Laurell. Lund Univ., Sweden.
B231
353
Flow Cytometric Sorting of Spermatozoa as a New Strategy to Improve the Quality of Spermatozoa in Assisted Reproductive Medicine S. Forte, G. Sartorius, F. Pletscher, M. De Geyter, H. Zhang and C. De Geyter. Univ. Hosp. of Basel and Univ. of Basel.
B232
354
Resolution of FOXP3+ Treg Cells Is Facilitated by Use of CD6 and PerFix-nc Kit F. Monsonis, J. Tung, J. Quintana, C. Garcia Santana and W. Godfrey. Beckman Coulter.
B233
355
VersaComp Antibody Capture Beads — A Versatile and Advanced Compensation Bead Product W. Godfrey, L. Yang, M. Rogers and J. Tung. Beckman Coulter.
B234
356
Reasons to Stop Collecting List Mode Data M. Naivar. DarklingX LLC.
B235
357
Hepatobiliary Transporters in Drug Safety Assessment R. Morgan. Amgen, Thousand Oaks, CA.
B236
358
Novel Flow Cytometry Method for the Detection of Neutrophil Activation and Degranulation in Peripheral Human Whole Blood D. Polancec, M. Vrancic, S. Dupont, K. Oreskovic and V. Erakovic Haber. Fidelta , Croatia, Galapagos SASU, France and Univ. of Rijeka, Croatia.
B237
359
An LED Pulser for Standardized Measurements of Fluorescence and Simplified Panel Development S. Perfetto, P. Chattopadhyay, R. Nguyen, D. Ambrozak, M. KuKuruga, M. Roederer and J. Wood. NIAID, NIH, USDA, Bethesda and Wake Forest Univ.
B238
360
Dual-Color Dynamic Live Cell-Based Assays for Autophagy C. Chen, T. Gaige, I. Zubonja and P. Hung. EMD Millipore.
B239
361
The S3™ Prodrop™ System, a Novel Approach to Automated Drop Delay Measurement and Monitoring C. Oxford, K. Kroeger, M. Ma, A. Vandergaw, D. Fox and S. Hunter. Bio-Rad Labs. and Propel Labs.
B240
362
4D Analysis of Akt Signaling in Breast Cancer K. Chin, M. Nederlof, M. CrapsterPregont, S. Kwon, S. Boddapati, K. Iljin, D. Sudar, R. Baehner, E. Barklis and J. Gray. Oregon Hlth. & Sci. Univ., UCSF, Quatitative Imaging Systs. LLC and Lawrence Berkeley Natl. Lab.
B241
363
Reduced Sample Loss by Remedial Engineering of BD Fortessa and LSR II Analyzers J. Hendrikx, M. Kweens, T. Baumgartner and C. O’Donnell. Mem. Sloan-Kettering Cancer Ctr.
ISAC 2013 Program and Abstracts
Speaker/Author Index
Multi-step Quality Control for a Multidimensional Cytometric Data Set of Clinical Importance S. Melzer, J. Bocsi, A. Szabó, A. Mittag, I. Dähnert and A. Tárnok. Univ. of Leipzig.
Poster Session Abstracts
351
Oral Session Abstracts
B229
Commercial Tutorials & Exhibits
ZAP-70 and IgVH Mutation Status in Indian Patients with Chronic Lymphocytic Leukemia R. Gupta, L. Rani, N. Mathur, S. Vishnubhatla, A. Sharma, L. Kumar and V. Raina. All India Inst. of Med. Sci.
Poster Session
350
Wednesday, 22 May
B228
Tuesday, 21 May
Cytomics – Importance of Multimodal Analysis of Cell Function and Proliferation in Biomonitoring M. Boumhras, B. Nasser, T. Nury, M. Cherkaoui-Malki and G. Lizard. Univ. of Hassan I Fac. of Sci. and Technol., Morocco and Univ. of Bourgogne, Dijon, France.
Monday, 20 May
349
Sunday, 19 May
B227
Saturday, 18 May
Assessment of Neutrophil Activation Antigen Up-Regulation in Whole Blood on the BD FACSVerse™ Cytometer Y. Zeng, B.R. Lee, M. Paulsen, J. Thorpe, L. Zhu and J.H. Allaert. BD Biosciences.
Special Lectures
348
Congress Overview
B226
63
Congress Overview
364
High Frequency of T Memory Stem Cells Precedes T Cell Immune-Reconstitution Following Human Bone Marrow Transplantation E. Lugli, A. Roberto, L. Castagna, S. Gandolfi, M. Roederer and D. Mavilio. Humanitas Clin. and Res. Ctr., Milan and NIAID, NIH.
Special Lectures
B243
365
A New, Automated Approach to Quality Control of High-Throughput Flow Cytometry Data P. Chattopadhyay and C. Fletez-Brant. NIAID, NIH.
B244
366
Genedata Screener Cell Population Extension: Interactive Processing of Single-Cell Data for Plate-Based High-Throughput Flow Cytometry J. Tupy, P. Aiello and O. Leven. Genedata, Switzerland.
B245
367
MoFlo Astrios Enhanced Forward Scatter: Simultaneous Sorting of Micro- and Macro-Samples A. Dean, C. Ross, B. McCarty and R. Morris. Beckman Coulter Life Science.
Sunday, 19 May
B246
368
Flow Cytometric Measurement of the CD11b Activated Epitope Expression on Human Neutrophils in Whole Blood as a Tool in Drug Development Process D. Polancec, M. Vrancic, S. Dupont, K. Oreskovic and V. Erakovic Haber. Fidelta, Croatia, Galapagos SASU, France and Univ. of Rijeka, Croatia.
Monday, 20 May
B247
369
Systematic Screening of Ovarian Cancer Cell Lines by Multicolour Flow Cytometry Reveals Tremendous Heterogeneity S. Sopper, M. Bösch, A. Zeimet, D. Reimer and D. Wolf. Med. Univ. Innsbruck and Univ. Clin. Bonn.
B248
370
The New Flow Cytometry Research Group of the Association of Biomolecular Resource Facilities A. Bergeron, A. Box, S. Chittur, M. Cochran, M. DeLay, P. Lopez, M. Meyer, T. Neubert, H. Pletcher and S. Tighe. Geisel Sch. of Med. at Dartmouth, Stowers Inst. for Med. Res., Univ. at Albany SUNY, Univ. of Rochester Med. Ctr., Cincinnati Children’s Hosp. Med. Ctr., NYU Ofc. of Collaborative Sci., Univ. of Pittsburgh, NYU Sch. of Med. and Univ. of Pennsylvania.
B249
371
New Method for High-Throughput Analysis and Sorting of Live Intact Plant Protoplasts R. Pulak and M. Malinouski. Union Biometrica Inc.
B250
372
Nine Color Flow Cytometric Characterization of Disease Progression in MRL/MpJFaslpr/j Mice W. Schott, D. Schultz, A. O’Neill, S. Grindle, E. Taylor, K. Snow and M. Strobel. The Jackson Laboratory.
B251
373
Using Imaging Flow Cytometry and Fish-In Suspension Method to Enumerate Human X Chromosomes by PCR-Generated Fluorescent Probes W. Wojciechowski and T. Bushnell. Univ. of Rochester Med. Ctr.
B252
374
High-Resolution Flow Cytometric Analysis of Human Immune Populations by a Competitive Clustering Algorithm T. Mosmann, J. Rebhahn, I. Naim, J. Cavenaugh, G. Sharma, J. Weaver, D. Roumanes and S. Quataert. Univ. of Rochester Med. Ctr. and Univ. of Rochester.
B253
375
FluoroFinder.com: A Free Flow Cytometry Resource to Simplify Polychromatic Panel Design R. Marcus, T. Powell, B. O’Connor, M. D’Souza, D. Mack, G. Veltri, J. Lannigan, S. Coquery and B. Palmer. FluoroFinder LLC, Univ. of Colorado Denver Sch. of Med., Natl. Jewish Hlth. and Univ. of Virginia Sch. of Med.
B254
376
Unsolved Challenges in Acoustic Focusing of Nanoparticles M.E. Piyasena and S.W. Graves. Univ. of New Mexico.
B255
377
Sorter Interface for High Resolution Confocal Imaging and Micro-Scale Live-Cell Matrix Experiments A. Donnenberg, V.S. Donnenberg, E.M. Meyer and C. Baty. Univ. of Pittsburgh Sch. of Med. and Univ. of Pittsburgh.
Speaker/Author Index
Poster Session Abstracts
Oral Session Abstracts
Commercial Tutorials & Exhibits
Poster Session
Wednesday, 22 May
Tuesday, 21 May
Saturday, 18 May
B242
64
ISAC 2013 Program and Abstracts
B257
379
Microfluidic Cell Sorters Enabled by Standing Surface Acoustic Waves T.J. Huang. Penn State.
B258
380
Parasite-Induced Immune Modulation of Dendritic Cells Identified Using Flow Cytometry J. Roberts, I. Sutherland, R. Shaw and A. Pernthaner. Hopkirk Res. Inst., AgResearch, New Zealand.
Saturday, 18 May
Evaluation of Flow Cytometry High-Throughput Cell Cycle Analysis on the Analyzers BD LSR Fortessa, BD Accuri C6, and the BD FACS Canto II D. Kratochwil-Otto, R. Maranchuk and A. Holme. Fac. of Med. and Dent., Univ. of Alberta.
Special Lectures
378
Congress Overview
B256
Sunday, 19 May Monday, 20 May Tuesday, 21 May Wednesday, 22 May Poster Session Commercial Tutorials & Exhibits Oral Session Abstracts Poster Session Abstracts Speaker/Author Index
ISAC 2013 Program and Abstracts
65
Congress Overview
COMMERCIAL TUTORIALS
Special Lectures
Sunday, 19 May Cost Effective Options for Optimizing Your Lab
Sunday, 19 May
Saturday, 18 May
Cytek Development 4059 Clipper Court Fremont, CA 94538 Phone: 804-234-8110 Email:
[email protected] Web: www.cytekdev.com 1245 – 1345 - Room 33AB Presenter: Lisa Nichols, PhD
Beckman Coulter Life Sciences 250 S. Kraemer Boulevard Brea, CA 92821 Phone: 714-342-9072 Email:
[email protected] Web: www.beckmancoulter.com 1245 – 1345 - Room 32AB Presenter: Mathias Streitz Michael Kapinsky Objective Flow Cytometry is a key component in immune monitoring. The flexibility and open nature of this technology makes comparison of results a major challenge in multicenter clinical studies such as the international trial conducted by The ONE Study consortium (www.onestudy.org). In order to assure consistency of results across participating laboratories, flow cytometry panel design and sample preparation procedures were designed to meet distinct requirements of robustness and reproducibility. Methods Multicolor panels, containing up to 9 colors, were constructed based on a set of rules related to optical properties, such as dye selection, based on brightness and emission spectra as well as taking the various types of known expression patterns into account. Conclusions The ONE Study panels could generally be useful for all laboratories performing immune monitoring for either multi-centre or local clinical trials. Multicolor panel construction needs to take into consideration the entire spectral and biological scenario related to a given panel, instead of selecting antibody-fluorochrome combinations based on single conjugate properties.
Speaker/Author Index
Poster Session Abstracts
Oral Session Abstracts
Commercial Tutorials & Exhibits
Poster Session
Wednesday, 22 May
Tuesday, 21 May
Monday, 20 May
What quality control measures can be used to ensure that cytometers are performing optimally? And, what options exist to expand your lab’s cytometry capabilities in the most cost-effective manner? Cytek Development is an established company capable of providing quality service and upgrades that optimize cytometers in order to keep pace with today’s experiments. This workshop introduces our latest offerings including a cross-platform application for evaluating cytometer performance and a new addition to our menu of custom upgrade options. Cytek’s Q&b method offers the ability to quantify performance on a variety of cytometers, provides absolute Q and b measurements (instead of relative), and calculates the resolution limit for each channel in MEFL units. We will discuss the relative advantages of the various Q and b cytometer validation programs. Cytek has also expanded its custom upgrade options for BD cytometers. We have sourced a compact 20mW 355 nm CW UV laser that is equal in performance to other typically used lasers. The compact size of this new laser allows it to be installed in FACSCalibur and LSR II models. We have integrated this option into our upgrades and will present data demonstrating their application.
The ONE Study – Novel Approaches in Multicolor Panel Design and Intracellular Preparation Chemistry
66
ISAC 2013 Program and Abstracts
Presenter: Kamala Tyagarajan, PhD
Beckman Coulter Life Sciences 250 S. Kraemer Boulevard Brea, CA 92821 Phone: 714-342-9072 Email:
[email protected] Web: www.beckmancoulter.com
Poster Session
1245 – 1345 - Room 29 CD Presenters: Vasilis Toxavidis John Tigges Dr. Robert Sleiman, Mr. Timothy Reed
Commercial Tutorials & Exhibits
There is great interest in both medical and scientific communities in submicron cell-derived particles, termed microparticles or microvesicles. Examples of tissues thatshed fragments of their plasma membrane (or microparticles) into the circulation include platelets, endothelial cells, leukocytes, and erythrocytes. There is increasing evidence that these submicron fragments have important physiological roles. Although many techniques have been used with limited success for the identification and characterization of microparticles, flow cytometry remains the most accurate and reproducible.
Oral Session Abstracts Poster Session Abstracts Speaker/Author Index
ISAC 2013 Program and Abstracts
MoFlo AstriosEQ – The New Standard in Forward Scatter and Fluorescence Dynamic Range Performance: A Case Study on Microparticles
Wednesday, 22 May
The Muse™ Cell Analyzer is a compact, affordable, revolutionary cell analyzer based on microcapillary cytometry that greatly simplifies cytometric analysis by using a guided touch screen interface and dedicated software modules to provide quantitative cellular data. The workshop will discuss the novel miniaturized flow technology utilized in the Muse™ Cell Analyzer, and also showcase data on the expanding range of optimized Muse™ assays available. A wide range of cell health assays allows for the rapid, single-step measurement of cell count and viability, provide information on cell cycle distribution and allow for characterization of apoptosis and death using Annexin V binding, caspase activity or mitochondrial depolarization. Immunology assays on the Muse™ platform allow for the identification and enumeration of CD4 T cells, CD8 T cells or B cells in whole blood or PBMC samples and also allow for obtaining information on activation status of lymphocytes based on CD69 or CD25 expression levels. Data from recent Cell Signalling assays on the platform will also be discussed. The combination of easy to perform assays with the simple, dynamic interface of the Muse™ Cell Analyzer can greatly empower researchers to obtain cytometric data with ease.
Designed for the detection of up to three RNA transcripts using flow cytometry, QuantiGene® Flow RNA Assay is an in situ hybridization assay that offers robust detection of RNA in individual cells and retains compatibility with antibody surface staining for simultaneous detection of protein. Incorporating dual oligonucleotide probe design with branched DNA signal amplification, this novel chemistry provides unique transcript expression in specific cell populations to develop biosignature profiles highly applicable in studying immune response.
Tuesday, 21 May
1245 – 1345 - Room 30CDE
Presenter: Sue Reynolds
Monday, 20 May
EMD Millipore 17 Cherry Hill Drive Danvers, MA 01932 Phone: 1-800-645-5476 Email:
[email protected] Web: www.emdmillipore.com
1245 – 1345 - Room 30AB
Sunday, 19 May
The Muse™ Cell Analyzer: A Versatile Platform for Simplified Cellular Analysis
eBioscience, an Affymetrix company 10255 Science Center Drive San Diego, CA 92121 Phone: 888-999-1371 Email:
[email protected] Web: www.ebioscience.com
Saturday, 18 May
1245 – 1345 - Room 31ABC
A Novel RNA ISH Assay for Flow Cytometry
Special Lectures
Sony Biotechnology, Inc. 2100 South Oak Street Champaign, IL 61820 Phone: 217-328-9396 Fax: 217-328-6292 Email:
[email protected] Web: www.sonybiotechnology.com
Congress Overview
Biosafe Sorting Solutions from Sony Biotechnology for Better, Faster, Easier Cell Sorting
67
Congress Overview
Traditionally however, the most challenging element has been the availability of sufficient forward scatter (FSC) resolution to delineate target populations from background signal and instrument noise.
Sunday, 19 May
Saturday, 18 May
Special Lectures
The Beckman Coulter new MoFlo AstriosEQ (AstriosEQ) sets a new standard in forward scatter and fluorescence dynamic range performance for cell sorting – delivering enhanced functionalityand sensitivity. Accommodating as standard, a 488 nm 200 mW diode laser with a conditioned flat-top shape beam profile, the AstriosEQ offers a patent pending FSC assembly capable of discriminating 200 nm to 30 µm particles on the same dynamic range. Designed for researchers who desire high productivity with more analytical capability, the AstriosEQ combines nano-scatter resolution with 5 decade fluorescence sensitivity on a 4-9 decade scale for the detection of submicron cell-derived particles.
Fluidigm 7000 Shoreline Ct. S. San Francisco, CA 94080 Phone: 650-266-6033 Web: www.fluidigm.com 12:45 – 13:45 - Room 29 AB
Tuesday, 21 May
Monday, 20 May
Therefore, we propose a patent pending innovative Forward Scatter detection module that will not only improve accuracy, but also allow for validation of the process by recovery of microparticles. The current study will present independent testing of the AstriosEQ demonstrating use of its two-parameter head-on PMT-based FSC optical assembly technology to identify and recover microparticles for downstream analysis.
Speaker/Author Index
Poster Session Abstracts
Oral Session Abstracts
Commercial Tutorials & Exhibits
Poster Session
Wednesday, 22 May
For Research Use Only. Not for use in diagnostic procedures.
68
ISAC 2013 Program and Abstracts
Congress Overview
Tuesday, 21 May
Union Biometrica, Inc. 84 October Hill Road Holliston, MA 01746 Phone: 508-893-3115 Fax: 508-893-8044 Email:
[email protected] Web: www.unionbio.com 1215 – 1315 - Room 32AB
Presenters: Brice Gaudilliere Gabi Fragiadakis
Presenter: Rock Pulak
Wednesday, 22 May Poster Session Commercial Tutorials & Exhibits Oral Session Abstracts Poster Session Abstracts
This tutorial demonstrates that the CyTOF can be used to detect dynamic immune changes in a clinically relevant setting. Mass cytometry-based immune monitoring of patients undergoing surgery thus holds the promise to identify patients at risk for aversive perioperative outcomes and to develop strategies to improve surgical recovery. These efforts may be particularly relevant in patients with advanced diseases (e.g. cancer), surgeries with high complication rates (e.g. infection after colo-rectal surgery), or specific vulnerabilities (e.g. extreme of ages).
Tuesday, 21 May
The tutorial will focus on the optimization of experimental conditions for metal-labeled antibody staining with the CyTOF® system, and data analysis of endogenous intracellular signaling activity in patients’ samples using Cytobank, a platform specifically designed for the analysis of both fluorescence and mass cytometry data, and SPADE, a hierarchical clustering algorithm that enables the visualization of signaling events across the hematopoietic continuum. Finally we will describe how these methods enabled the system-wide analysis of endogenous signaling events in patients undergoing surgery.
Union Biometrica extends the utility of flow cytometry to samples that are too large or too fragile for traditional flow instruments. This technology provides a method for analysis and dispensing, increasing throughput and replacing manual sorting. The BioSorter™ flow cytometer is designed to provide the flexibility for working with samples ranging in sizes from 1 to 1500 microns. This very broad range of sizes includes many interesting types of cells, cell clusters and even whole organisms. We present some new applications of the BioSorter, including the use of large delicate cells such as adipocytes and plant protoplasts, cell clusters such as mammalian neurospheres, plant calli and fungal pellets, and cells encapsulated within hydrogel particles. These samples can be analyzed and dispensed intact owing to the low sample pressures, low shear forces and the gentle sorting mechanism. Union Biometrica has developed an autosampler, LP Sampler™ that automatically loads samples from multiwell plates to the BioSorter for analysis and resorting, and also the VAST BioImager, a simplified flow-based platform for image acquisition and analysis. Union Biometrica continues to develop research tools for large particle analysis.
Monday, 20 May
A critical component of immune monitoring is analyzing the dynamics of the immune system in response to perturbation. This tutorial examines the phenotypic and signaling single-cell responses in patients undergoing surgery, a clinically relevant immune perturbation. Using mass cytometry we are able to characterize this response at the systems level with single-cell resolution.
Sunday, 19 May
1215 – 1315 - Room 33AB
Saturday, 18 May
DVS Sciences 639 N. Pastoria Avenue Sunnyvale, CA 94085 Phone: 408-900-7224 Email:
[email protected] Web: www.dvssciences.com
Flow Cytometry for Large Delicate Cells, Cell Clusters, Encapsulated Cells, and Small Model Organism
Special Lectures
Mass Cytometry Reveals an Endogenous Immune Response to Physiological Perturbation in Humans – Optimization and Design
Speaker/Author Index
ISAC 2013 Program and Abstracts
69
Congress Overview
Biosafety and Sorting Solutions from BD Biosciences
Saturday, 18 May
Special Lectures
BD Biosciences 2350 Qume Drive San Jose, CA 95131 Phone: 1-877-232-8995 Email:
[email protected] Web: www.bdbiosciences.com 1215 – 1315 - Room 31ABC Presenters: Gil Reinin Janet Horta
Wednesday, 22 May
Tuesday, 21 May
Monday, 20 May
Sunday, 19 May
In the last few years there has been an increased customer awareness of the biosafety risk from aerosols produced in a clog situation while cell sorting, especially when sorting unfixed human cells. This concern was validated by aerosol generation studies where the data demonstrated that aerosol droplets produced from a stream clog could potentially be a safety risk if the proper biosafety measures were not taken. Biosafety measures include a proper risk assessment of samples being run, wearing proper PPE, always using an aerosol management system, and if required, the use of a BSC that can pass international biosafety standards such as the NSF-49 standard for product, personnel, and environmental protection. As a result of these guidelines a greater percentage of sorters are being purchased with biosafety cabinets. In this seminar we will review recent biosafety guidelines for sorting and discuss solutions for meeting these guidelines for BD Biosciences sorters.
Commercial Tutorials & Exhibits
Poster Session
Innovative Spectral Analysis Technology and Novel Applications for Cellular Analysis from Sony Biotechnology
FlowJo Ninja Skills
Tree Star, Inc. 340 A Street Suite 101 Ashland, OR 97520 Phone: 800-366-6045 Fax: 541-482-3153 Email:
[email protected] Web: www.flowjo.com
1215 – 1315 - Room 30AB Presenters: Nicholas Ostrout, PhD John Quinn, PhD Become a FlowJo Ninja! Many users aren’t aware of all the amazing features, interesting short cuts, and awesome options to help improve and streamline your data analysis in FlowJo. Now with version 10 of FlowJo, this list has grown even longer! Come hone your data analysis skills, learn new techniques, and become a master of level 10 FlowJo. Of course becoming a first degree ninja shouldn’t be enough, so earn your second degree and stay around for Fluorish. We will be showcasing the redesigned panel builder! Do you have what it takes to be a master panel designer? If it has been a while since you last saw Fluorish, the website and panel builder now have a growing list of new features that will make your life (ok, maybe just your life in flow cytometry) much easier. Quickly locate reagents from more than 10 major antibody suppliers, manage a core, grab the instrument configurations, create a Fluorish lab to share/manage your lab inventory, and order everything you need for your flow experiments from one spot.
Oral Session Abstracts
Sony Biotechnology, Inc. 2100 South Oak Street Champaign, IL 61820 Phone: 217-328-9396 Fax: 217-328-6292 Email:
[email protected] Web: www.sonybiotechnology.com
Speaker/Author Index
Poster Session Abstracts
1215 – 1315 - Room 30CDE
70
ISAC 2013 Program and Abstracts
Presenter: TBD Bio-Rad presents the all new S3(™) Cell Sorter. In partnership with Propel Labs, the S3 cell sorter makes cell sorting easy to use and accessible to researchers with several automated features. Using state of the art technology, ProDrop(™) automates the drop delay procedure providing accurate and consistent droplet deflection. The S3 cell sorter is fully featured with an onboard temperature control using Peltier solid state system, completely enclosed fluidic and a convenient, compact space saving design- all without compromising in performance or sensitivity. Created with researchers in mind, an affordable 1-4 fluorescent parameter system and equipped with up to two lasers, this system is designed to accommodate everyday experiments amongst cell biologist. Complimentary to systems currently in flow core labs, offload 1-4 color experiments onto the S3 cell sorter that researchers can use on their own with minimal training.
Monday, 20 May Tuesday, 21 May Wednesday, 22 May Poster Session Commercial Tutorials & Exhibits Oral Session Abstracts
Optimization of a novel microchip-based cell sorting valve has recently been combined with flow cytometry analytic capabilities to create a novel closed cell sorting platform. The microchip-based valve is a MEMS (Micro Electro Mechanical System) that delivers the robustness, precision and speed of silicon chip products combined with a magnetic actuation mechanism for high-speed cell sorting. The system uses typical fluorescence gating selection logic to actuate the valve which directs cells to one of two microfluidic paths in a closed single-use cartridge. Thus, in a typical workflow, an input sample containing 10e7-9 cells is processed into a sample highly enriched for the desired cell population(s) and a sample depleted for such cells. All the cells remain in the cartridge and can be recovered for subsequent analysis or processing, including re-sorting. The cells remain in buffer or media undiluted by sheath, and cell viability appears unchanged by the sorting process. The purity of the recovered sort sample depends on the input cell density and the frequency of desired cells as predicted by Poisson statistics. Characterization of the platform’s performance will be presented in the context of several relevant cell purification protocols including isolation of human T regulatory cells (T regs), tetramer-positive human T cells and circulating tumor cells (CTCs) from peripheral blood in an experimental murine model of pancreatic ductal carcinoma.
1215 – 1315 - Room 29AB
Sunday, 19 May
Presenters: John Foster, President, Owl biomedical, Inc. Jack Dunne, Executive Vice President, Science & Technology, Owl biomedical, Inc.
Bio-Rad 2000 Alfred Nobel Hercules, CA 94547 Phone: 510-741-4494 Email:
[email protected] Web: www.bio-rad.com
Saturday, 18 May
1215 – 1315 - Room 29CD
The S3 Cell Sorter – Cell Sorting Made Simple
Special Lectures
Miltenyi Biotec GmbH Friedrich-Ebert-Strasse 68 51429 Bergisch Gladbach Germany Phone: 49 2204 83060 Email:
[email protected] Web: www.miltenyibiotec.com
Congress Overview
Fluorescence-Activated Cell Sorting without Droplets
Poster Session Abstracts Speaker/Author Index
ISAC 2013 Program and Abstracts
71
Congress Overview
Wednesday, 22 May
Special Lectures
Studies of T-Cell/Antigen Presenting Cell Conjugation Using Imaging Flow Cytometry
Saturday, 18 May
EMD Millipore 17 Cherry Hill Drive Danvers, MA 01932 Phone: 1-800-645-5476 Email:
[email protected] Web: www.emdmillipore.com
Sunday, 19 May
1215 – 1315 - Room 33AB Presenter: Haley Pugsley, PhD
Molecular Devices LLC 1311 Orleans Drive Sunnyvale, CA Phone: 1-800-635-5577 Email:
[email protected] Web: www.moleculardevices.com 1215 – 1315 - Room 32AB Presenter: Evan F. Cromwell, PhD, Director of Assay Development A continuing trend in drug discovery and development is the use of complex cell-based models that are able to provide more biologically relevant & predicative assays. We present new advancements in plate reader and imaging cytometry systems that enable multiplexed analysis of important biological outputs. The system has been designed for environments from basic research labs to medium/ high throughput screening cores, and the intuitive user interface allows non-imaging specialists to get up and running quickly. We will present examples of use of the system for cells-based assays including plate QC and models for testing anti- inflammatory compounds and in vitro toxicity assessment.
Speaker/Author Index
Poster Session Abstracts
Oral Session Abstracts
Commercial Tutorials & Exhibits
Poster Session
Wednesday, 22 May
Tuesday, 21 May
Monday, 20 May
Imaging flow cytometry combines the speed, sensitivity, and phenotyping abilities of flow cytometry with the detailed imagery and functional insights of microscopy. This unique combination enables a broad range of applications that would be impossible using either technique alone. This seminar will explore two studies of the T-cell/Antigen Presenting Cell (APC) Immune Synapse performed on the Amnis® FlowSight® and ImageStreamX Mark II imaging flow cytometers. In the first study we will demonstrate image-based parameters that were used to assess the frequency of T-cell/APC conjugates with an organized immune synapse in an objective and statistically significant manner. In the second study we evaluate the specific location of the adhesion and signaling molecules LFA-1 and Lck within the immunological synapse complex in T-cells when presented with SEB, as well as the activation of the T cells in contact with APC and with organized immunological synapse by measuring the nuclear localization of NFkB in the T cell.
Complex Cell Based Assays Using SpectraMax® i3 Multi-Mode Plate Reader and SpectraMax® MiniMax™ Imaging Cytometer
72
ISAC 2013 Program and Abstracts
Taking Flow Cytometry to the Limits PARTEC Am Flugplatz 13 Goerlitz Saxony, Germany 02828 Phone: 49 0 3581 87460 Fax: 49 0 3581 874670 Email:
[email protected] Web: www.partec.com
Presenters: Dr. Roy Overton Dr. Danny Koehler
Advances in technology have made multi-color flow cytometry more accessible to researchers worldwide.
Taking flow cytometry to the limits - Partec Nano/ ViroFlow™ & Nano/ViroSort™: Detection and sorting of viruses and other nanoparticles. - Sorting neurons, cardiomyocytes and other fragile cells gently and safely with the Partec CyFlow® Sorter. - Highresolution DNA analysis.
Today scientists choose dyes based on the capabilities of the instrumentation available, the number of surface receptors on the cells they are studying, their brightness and spillover value.
• How the impact of optical design of the cytometer on signal detection
Verity Software House 45A Augusta Road PO Box 247 Topsham, ME 04086 Phone: 207-729-6767 Fax: 207-729-5443 Email:
[email protected] Web: www.vsh.com 1215 – 1315 - Room 30AB
• Importance of standardization of instruments and optimization of instrument setup to data quality
Presenters: Dr. Beth Hill Mark Munson
• Importance of the use of appropriate controls
Poster Session
• Methodology for choosing fluorochromes for optimum multicolor panels – with data from 6 to 18 colors
Wednesday, 22 May
Topics include
GemStone: High-Dimensional Analysis Principles and Applications
Tuesday, 21 May
The tutorial will present a methodology for designing multicolor experiments, including demonstrations of how effective use of fluorochromes allows for the identification of cell populations with lower receptor density than was previously possible. A wide range of data using this methodology will be presented.
Monday, 20 May
Presenter: Robert Balderas, Vice President, Biological Sciences
Sunday, 19 May
1215 – 1315 - Room 30CDE
Saturday, 18 May
1215 – 1315 - Room 31ABC
Special Lectures
BD Biosciences 2350 Qume Drive San Jose, CA 95131 Phone: 1-877-232-8995 Email:
[email protected] Web: www.bdbiosciences.com
Congress Overview
Advances in Technology Have Made Multi-color Flow Cytometry More Accessible to Researchers Worldwide
Commercial Tutorials & Exhibits
This first part of this presentation is designed to introduce the principles of multi-dimensional data modeling via a common example, with a closing presentation on current applications. The unique capabilities of GemStone’s Probability State Modeling will be presented in a familiar format, with perhaps an unfamiliar twist. There will be opportunities for discussion, questions, and review.
Oral Session Abstracts Poster Session Abstracts Speaker/Author Index
ISAC 2013 Program and Abstracts
73
Congress Overview Special Lectures
The Latest Advances in Cytometry Powered by Molecular Probes® Life Technologies™ Presents: Discovery Using the Attune® Acoustic Focusing Cytometer, Molecular Probes® Reagents, and Evos® Cell Imaging Sytems
Sunday, 19 May
Saturday, 18 May
Life Technologies 5791 Van Allen Way Carlsbad, CA 92008 Phone: 760-603-7200 Email: technical-molecularprobes@lifetech. com Web: www.probes.com 1215 – 1315 - Room 29CD
Monday, 20 May
Presenter: Julie K. Jang, Department of Pathology, Keck School of Medicine, University of Southern California
De Novo Software 3250 Wilshire Boulevard, Suite 803 Los Angeles, CA 90010 Phone: 213-814-1240 Fax: 213-814-1511 Web: www.denovosoftware.com 1215 – 1315 – Room 29AB Presenters: David Novo (De Novo Software) and Peter Li (Nexcelom Biosciences) Digital Microscopy and Image Cytometry are rapidly evolving technologies in biomedical research and drug discovery. However, with imaging, managing your analysis results and images is a daunting task. Nexcelom’s image cytometry solutions automate the time-consuming counting procedures for hundreds of different cell types, enabling scientists to focus less on the process and more on the research results. Nexcelom’s products are currently being used in the labs of leading pharmaceutical companies, Biotech organizations, universities, and research institutions. By combining the power of image cytometry with De Novo Software’s FCS Express 4 Cytometry software you can now manage your multi-parametric data image cytometry data sets with a proven flow cytometry styled analysis. With FCS Express 4 Cytometry you combine data processing and image analysis with report building in a single package. Eliminate the redundant data processing steps you now perform in Excel, and create PowerPoint presentation with a click of a button. Work with your images and multi-parametric data dynamically in FCS Express and then simply export a report in the format you desire. Nexcelom’s Vision CBA is now compatible with FCS Express Cytometry to provide users a unique analysis solution for simple to complex image analysis experiments. Join the Nexcelom Biosciences and De Novo Software teams to learn how to turn your image cytometry data into results.
Speaker/Author Index
Poster Session Abstracts
Oral Session Abstracts
Commercial Tutorials & Exhibits
Poster Session
Wednesday, 22 May
Tuesday, 21 May
Life Technologies’™ Attune® Acoustic Focusing Cytometer is the first-of-its-kind cytometry system to use sound waves to precisely control the focusing of particles and cells. The same scientists who designed and built Attune® inspire true discovery in flow cytometry with the newest flow cytometry reagents and selection tools from Molecular Probes® - reagents and tools for flow cytometry that take significant steps forward in the analysis of cellular function, far beyond immunophenotyping. The EVOS® line of Cell Imaging Systems are designed to work together – from the initial cell culture check (for viability and morphology) to more complex analyses such as time lapse, image tiling and stitching. The EVOS® systems make cell imaging accessible to almost every lab application and budget. Molecular Probes® integrated solutions will help you design and execute your flow cytometry and cell imaging experiments. Late breaking data from both platforms will be presented.
Getting the Most Out of Your Image Cytometry Data with the Nexcelom Vision CBA and FCS Express Cytometry
74
ISAC 2013 Program and Abstracts
Congress Overview
EXHIBITOR SHOWCASE Monday, 20 May
Special Lectures
Exhibit Hall GH
Novel Polymer Fluorochromes and their Application in Multicolor Flow Cytometry
Large, Multidimensional Data Sets Analysed in Seconds!
1215 – 1225 - Exhibit Hall
1255 – 1305 - Exhibit Hall
Polymer dye technology from Sirigen, a BD Biosciences company, has opened the door to the development of novel dyes that are four to ten times brighter than conventional dyes, with equivalent background — a breakthrough in the field. For cells that have few receptors on the surface, bright reagents are essential in resolving these dim cells from others in a sample. The characteristics of the Sirigen polymer dyes enable them to achieve much brighter fluorescence signals than traditional organic fluorescent dyes or even phycobiliproteins such as PE or APC. This means that they are effective for identifying cell populations with a broader range of receptor density than previously possible. Come learn about the latest advances in polymer dye development from BD Biosciences.
SOPHE represents a breakthrough in processing flow cytometer generated data sets.
• Analyse large data sets with 20 + parameters or dimensions and hundreds of thousand cells. • Divide the data into clusters without requiring prior knowledge of the number of clusters. The SOPHE algorithms find these otherwise hidden relationships and report the characteristics of each such cluster. The SOPHE method delivers results in a few seconds. The algorithms on which SOPHE is based are patent protected by PCT/AU2012/000252 “Multidimensional cluster analysis”
Poster Session Commercial Tutorials & Exhibits
handyem aims to democratize cytometry by providing day-to-day autonomy and agility to life science researchers with simple, highly efficient instruments at a fraction of the cost of today’s solutions. handyem’s HPC-100 Personal Cytometer, with its F3 microchip platform, brings simplicity, accessibility and ease of use to life science researchers and core lab managers. This short presentation of the HPC-100 will draw the main highlights of the solution.
The algorithms in SOPHE can: Wednesday, 22 May
1235 – 1245 - Exhibit Hall
Data sets generated can have 10 or even 20 independent dimensions, and each dimension in any combination can generate sets of clusters. Current software systems cannot do this level of cluster analysis, and where they attempt it, it is incomplete, slow, and computationally inefficient.
Tuesday, 21 May
Flow Cytometry in your Hands®
The development of flow cytometer hardware has been advancing faster than the ability to analyse the data generated by the instrumentation with its ever increasing number of variables or markers contained within the data sets.
Monday, 20 May
handyem
Sunday, 19 May
NewSouth Innovations Saturday, 18 May
BD Biosciences
A brief demonstration of the software will be presented and an opportunity for discussion with one of the key developers Dr John Zaunders.
Oral Session Abstracts
BioStatus DRAQ7™ - A Unique Far-Red Real-Time Viability Monitor
Poster Session Abstracts
1315 – 1325 - Exhibit Hall
ISAC 2013 Program and Abstracts
Speaker/Author Index
DRAQ7(tm) is a patented far-red fluorescing anthraquinone-derived viability dye that extends the opportunities for multi-colour flow cytometry and image-based assays. It allows facile combination with commonly used orange/red mitochondrial health probes for temporal interrogation of apoptosis and has been validated to permit new real-time viability monitoring. 75
Congress Overview Poster Session Abstracts
Oral Session Abstracts
Commercial Tutorials & Exhibits
Poster Session
Wednesday, 22 May
Tuesday, 21 May
Monday, 20 May
Sunday, 19 May
Saturday, 18 May
Special Lectures
Exhibitor Listing Company Booth(s)
Company Booth(s)
AAT Bioquest, Inc........................................... 625 Abcam............................................................ 614 ALPCO Diagnostics........................................ 715 Bangs Laboratories......................................... 612 Bay Bioscience Co., Ltd.................................. 501 BD Biosciences.............................................. 420 Beckman Coulter, Inc..................................... 602 BioLegend...................................................... 601 Bio-Rad Laboratories...................................... 619 Biostatus Limited............................................ 406 BioTek Instruments, Inc.................................. 909 Blue Sky Research.......................................... 438 Brooks Automation Ltd................................... 212 Caltag Medsystems......................................... 414 Cedarlane....................................................... 733 Chroma Technology Corp............................... 810 Cobolt............................................................ 629 Coherent, Inc.................................................. 620 Cytek Development, Inc................................. 313 Cytobank Inc.................................................. 442 CYTOGNOS................................................... 721 De Novo Software.......................................... 329 DVS Sciences Inc........................................... 530 eBioscience, an Affymetrix company.............. 723 EMD Millipore................................................ 814 Etaluma.......................................................... 222 EXBIO Praha a.s............................................. 538 Flow Contract Site Laboratory, LLC................. 404 Fluidigm......................................................... 631 GE Healthcare................................................ 808 Hamamatsu Corporation................................ 440 handyem........................................................ 719 Hellma USA, Inc............................................ 520 iLab Solutions................................................. 713 IMGENEX Corporation................................... 840
Innova Biosciences......................................... 502 IntelliCyt Corporation..................................... 907 ISAC............................................................... 742 Life Technologies............................................ 624 Market Tech.................................................... 213 Melles Griot................................................... 634 Miltenyi Biotec GmbH................................... 512 Molecular Devices, LLC................................. 802 Newport Corporation..................................... 504 NewSouth Innovations Pty Ltd........................ 323 Nexcelom Bioscience..................................... 333 Novus Biologicals........................................... 804 OSELA Inc...................................................... 605 OXXIUS Lasers............................................... 506 PARTEC.......................................................... 830 Pavilion Integration Corporation..................... 729 Phoenix Flow Systems, Inc............................. 630 Photop Technologies Inc................................. 211 Propel Labs..................................................... 623 Semrock......................................................... 632 Sony Biotechnology Inc.................................. 303 SouthernBiotech............................................. 311 Spherotech, Inc.............................................. 416 Stratedigm, Inc............................................... 522 Thermo Fisher Scientific................................. 633 Tonbo Biosciences.......................................... 513 Toptica Photonics Inc..................................... 505 Tree Star, Inc................................................... 611 TTP Labtech Ltd.............................................. 325 Union Biometrica, Inc.................................... 711 Verity Software House.................................... 402 Vortran Laser Technology, Inc......................... 319 Wiley............................................................. 842 Woodside Logic............................................. 321 Xitogen Technologies Inc........................ 516, 615
Exhibit Hours
Speaker/Author Index
Monday, 20 May Tuesday, 21 May Wednesday, 22 May
76
1130 – 1900 1130 – 1900 1130 – 1630
ISAC 2013 Program and Abstracts
Special Lectures
Saturday, 18 May
Sunday, 19 May
Monday, 20 May Tuesday, 21 May
Wednesday, 22 May
Poster Session
Commercial Tutorials & Exhibits
Oral Session Abstracts
Poster Session Abstracts
Speaker/Author Index
77
ISAC 2013 Program and Abstracts
Congress Overview
CYTO 2013 EXHIBITS
Monday – Wednesday, 20 – 22 May Exhibit Hall GH
Congress Overview
Exhibiting Companies
Special Lectures
Disclaimer
Saturday, 18 May
Participation in the Exhibits Program does not constitute an endorsement by the International Society for Advancement of Cytometry (ISAC) of the claims, products or services offered.
AAT Bioquest, Inc.
625
Sunday, 19 May
520 Mercury Drive Sunnyvale, CA 94085 Phone: 408-733-1055 Fax: 408-733-1304 Email:
[email protected] Web: www.aatbio.com
Monday, 20 May Tuesday, 21 May
1 Kendall Square, Suite B2304 Cambridge, MA 02139 Phone: 617-577-4277 Fax: 617-225-3938 Email:
[email protected] Web: www.abcam.com
Poster Session
Wednesday, 22 May
Abcam sells high-quality, research-grade antibodies and associated protein research tools. Comprehensive datasheets, together with expert customer support and fast delivery, continue to make Abcam the researcher’s choice. Abcam’s catalogue also includes a growing range of non-antibody products, such as proteins, peptides, lysates, immunoassays and other kits and biochemicals --- all available at www.abcam.com.
715
Oral Session Abstracts
Commercial Tutorials & Exhibits
26-G Keewaydin Drive Salem, NH 03079 Phone: 800-592-5726 Fax: 603-898-6854 Email:
[email protected] Web: www.alpco.com ALPCO is a provider of life science research tools for academic, pre-clinical and clinical use. Our comprehensive portfolio includes immunoassays for a variety of therapeutic areas of interest including Cytokines and Cell Signaling. We recently expanded our product catalog with the addition of Flow Cytometry reagents. Stop by our booth to learn more, or visit www.alpco.com.
Bangs Laboratories, Inc. produces fluorescence microsphere standards to help the research and clinical community conduct studies and evaluate data in an easier, more precise manner. Our products set the standard for quality control and quantitation in the fields of flow cytometry, fluorescence microscopy, image analysis, and other fluorescence analytical applications. Superior Customer and Technical Service complement Bangs’ extensive product offerings.
Bay Bioscience Co., Ltd.
501
2-5, Minatojima Minamimachi 5-chome, Chuo-ku Kobe, 650-0047 Japan Phone: 81 78 304 5881 Fax: 81 78 304 5889 Email:
[email protected] Web: www.baybio.co.jp Bay Bioscience designs, develops, and manufactures high performance cell sorting and analysis systems. The company’s current products, the JSAN and JSAN Swift combine high performance and affordability in a way previously not available. Configure a system to exactly suit your needs, including true UV excitation. Please visit the company website at www.baybio. co.jp The JSAN: desktop sorting at an affordable price. Categories Cell separation products Data analysis systems and software Flow cytometry and cell sorting equipment
Speaker/Author Index
Poster Session Abstracts
612
9025 Technology Drive Fishers, IN 46038 Phone: 317-570-7020 Fax: 317-570-7034 Email:
[email protected] Web: www.bangslabs.com
Abcam 614
ALPCO Diagnostics
Bangs Laboratories
78
ISAC 2013 Program and Abstracts
420
2000 Alfred Nobel Drive Hercules, CA 94547 Phone: 510-741-4494 Email:
[email protected] Web: www.bio-rad.com
100 Tigan Street Winooski, VT 05404 Phone: 802-655-4040 Fax: 802-655-7941 Web: www.biotek.com BioTek is a leader in the design, manufacture, and sale of microplate instrumentation and software. BioTek’s instrumentation, like the just-released Cytation3 Cell Imaging Multi-Mode Microplate Reader, a system that combines automated microscopy and Hybrid multimode detection, is used to aid in the advancement of life science research, facilitate the drug discovery
Poster Session Abstracts Speaker/Author Index
ISAC 2013 Program and Abstracts
909
Oral Session Abstracts
World-Class Antibodies, Proteins, Assays and Research Solutions. Complete Brilliant Violet™ Antibody Conjugates for the Violet Laser: BV510™, BV711™, BV785™. Personalized Multicolor Flow Cytometry Panel Design. New LEGENDScreen™ Human Cell Screening (PE) Kits. Request Bulk Cytokines & Chemokines for Bioassay. Ultra-LEAF™ (Low Endotoxin, Azide-Free) Antibodies. New ELISA Kits: IL-35, Active TGF-ß1.
BioTek Instruments, Inc.
Commercial Tutorials & Exhibits
9727 Pacific Heights Boulevard San Diego, CA 92121 Phone: 858-455-9588 Fax: 858-455-9587 Email:
[email protected] Web: www.biolegend.com
Our latest product - DRAQ7™ - is the far-red cell impermeant DNA dye DRAQ7™ only enters cells with compromised membranes: dead, damaged, and apoptotic cells for enumeration and sorting. DRAQ7™ is ready-to-use, permits multi-colour analysis and does not interfere with GFP, Annexin V and mitochondrial health probes (just like DRAQ5™!). DRAQ7™ shows negligible toxicity for use in longterm, real-time assays and can be used as a viability reporter in RNAi knock-downs and toxicity assays.
Poster Session
BioLegend 601
56 Charnwood Road, Shepshed Loughborough LEICS LE12 9NP United Kingdom Phone: 44 1509 558163 Fax: 44 1509 651061 Email:
[email protected] Web: www.biostatus.com
Wednesday, 22 May
Research Applications Offering the broadest range of cellular analysis systems in the world, Beckman Coulter provides a variety of reagents, instruments and software to meet the diverse needs of today’s research laboratories.
406
Tuesday, 21 May
Clinical Applications Beckman Coulter provides a comprehensive line of flow cytometry solutions that lead the charge against life’s most challenging diseases. Our instruments, sample preparation systems and family of antibodies offer an unparalleled continuum of cellular testing.
Biostatus Limited
Monday, 20 May
250 S. Kraemer Boulevard Brea, CA 92822 Phone: 714-961-3260 Web: www.beckmancoulter.com
Sunday, 19 May
602
Discover Bio-Rad’s automated, easy-to-use benchtop S3 cell sorter that is affordable to researchers. Bio-Rad continues to provide a wide variety of workflow solutions in instrumentation and reagents for flow cytometry, transfection, cell counting, droplet digital PCR, conventional and real-time PCR, amplification reagents and primers, cancer biomarkers, electrophoresis, blotting-systems, chromatography, and imaging.
Saturday, 18 May
BD Biosciences, a segment of Becton, Dickinson and Company, is one of the world’s leading businesses focused on bringing innovative tools to life science researchers and clinicians. Its product lines include: flow cytometers, cell imaging systems, monoclonal antibodies, research reagents, diagnostic assays, and tools to help grow tissue and cells.
Beckman Coulter, Inc.
619
Special Lectures
2350 Qume Dr. San Jose, CA 95131 Phone: 877-232-8995 Fax: 408-954-2009 Email:
[email protected] Web: www.bdbiosciences.com
Bio-Rad Laboratories
Congress Overview
BD Biosciences
79
Congress Overview Special Lectures
process, provide rapid, cost-effective industrial analysis and to enable sensitive and accurate quantification of a wide range of molecules across diverse applications.
Blue Sky Research
438
Saturday, 18 May
1537 Centre Pointe Drive Milpitas, CA 95035 Phone: 408-941-6003 Email:
[email protected] Web; www.blueskyresearch.com
Sunday, 19 May
Brooks Automation Ltd.
212
Monday, 20 May Wednesday, 22 May
Tuesday, 21 May
Brooks Life Science Systems provides a wide range of automated solutions including the Celigo®Imaging Cytometer. With both brightfield and fluorescence imaging capabilities, Celigo enables whole well automated, label-free cell counting, single cell detection and clonal expansion as well as spheroid analysis. Celigo’s fluorescence functionality provides rapid image acquisition and analysis. With unrivaled simplicity and ease of use, Celigo is the fastest imaging cytometer available today.
414
Commercial Tutorials & Exhibits
Poster Session
Whiteleaf Business Centre 11, Little Balmer Buckingham Buckinghamshire MK18 1TF United Kingdom Phone: 01280 827460 Email:
[email protected] Web: www.cytomark.co.uk/
Oral Session Abstracts
Caltag Medsystems is a UK based manufacturer and distributor of flow cytometry products. The Cytomark Division of Caltag MedSystems is a manufacturer of blood stabilisation products and stabilised whole Blood Controls. TransFix® - A Cellular Antigen Stabilisation Reagent. TransFix® is used to stabilise whole blood, plus Cerebrospinal Fluid (CSF), Bone Marrow and Fine Needle Aspirates. Cytomark whole Blood Controls are used to verify absolute cell counts and instrument set up.
810
10 Imtec Lane Bellows Falls, VT 05101 Phone: 802-428-2500 Fax: 802-428-2525 Email:
[email protected] Web: www.chroma.com Optical components provide precise color separation and signal purity for applications such as fluorescence microscopy, flow cytometry, confocal or multiphoton microscopy. BP/LP/SP * Multiband * Notch * Dichroic Mirrors * Polychroic Mirrors * UV/VIS/NIR * AR Coatings * Hot/Cold Mirrors * ND/AG/AL Mirrors * Laser Grade and more, engineered and manufactured by a team of employee-owners committed to bringing you the finest optical filters, filter sets and optics solutions. Chroma Technology, 800-824-7662.
Cobolt 629 Vretenvagen 13 SE-171 54 Solna Sweden Phone: 46 8 54591230 Fax: 46 8 54591231 Email:
[email protected] Web: www.cobolt.se Cobolt AB (Stockholm, Sweden) has, since year 2000, been committed to development and supply of innovative laser products that meet or exceed the market’s expectations concerning performance, quality and robustness. Through continuous technology development, customer orientation and an ISO-certified quality management system, Cobolt has become a preferred supplier of lasers to major manufacturers of analytical instrumentation equipment and leading research labs.
Speaker/Author Index
Poster Session Abstracts
1210 Turrentine Street Burlington, NC 27215 Phone: 336-513-5135 Fax: 336-513-5138 Email:
[email protected] Web: www.cedarlanelabs.com
Chroma Technology Corp
Northbank Irlam, Manchester M44 5AY United Kingdom Phone: 44 (0) 161 722-2000 Web: www.brooks.com
Caltag Medsystems
Cedarlane 733
80
ISAC 2013 Program and Abstracts
620
De Novo Software
442
639 N. Pastoria Avenue Sunnyvale, CA 94085 Phone: 408-900-7224 Fax: 408-900-7224 Email:
[email protected] Web: www.dvssciences.com
Poster Session Abstracts
DVS Sciences Inc. is an analytical equipment and reagents development company that produces and markets Instruments and reagents for mass cytometry. Multiplex analysis capability of the CyTOF® system enables new insights into the functional complexity of biological systems at the single cell level.
Speaker/Author Index
ISAC 2013 Program and Abstracts
530
Oral Session Abstracts
Cytobank Inc. provides scientific solutions built on Cytobank, a cloud-computing platform focused on single cell technologies. We are a team of scientist-entrepreneurs from Stanford University with expertise in cytometry, cell signaling, drug discovery, informatics and software development. We are the engine behind landmark papers in the growing field of high dimensional cytometry and have partnerships with key vendors in the field.
DVS Sciences Inc.
Commercial Tutorials & Exhibits
800 West El Camino Real, Suite 180 Mountain View, CA 94040 Phone: 650-265-7806 Fax: 650-204-6866 Email:
[email protected] Web: www.cytobank.org
De Novo Software specializes in producing high quality cytometry data analysis software. Our flagship product, FCS Express 4, is a full feature solution for Flow Cytometry data. FCS Express 4 Image Cytometry introduces the same support and flexibility for imaging data files. FCS Express combines a user friendly modern interface with powerful analysis tools, visualization capabilities and sophisticated presentation features, making it the tool of choice for thousands of researchers needing quick results from their data.
Poster Session
Cytobank Inc.
3250 Wilshire Boulevard, Suite 803 Los Angeles, CA 90010 Phone: 213-814-1240 Fax: 213-814-1240 Email:
[email protected] Web: www.denovosoftware.com
Wednesday, 22 May
Cytek provides the flow cytometry tools scientists need at prices they can afford accompanied by cost effective service programs. Customize a high-powered cytometer from a menu of industry standard platforms at a fraction of the cost of a new system. Or, select an upgrade package to expand the capabilities and extend the useful life of your existing cytometer. Finally, our comprehensive service plan options allow you to select coverage that aligns with your needs and resources.
329
Tuesday, 21 May
4059 Clipper Court Fremont, CA 94538 Phone: 510-657-0102 Fax: 510-657-0151 Email:
[email protected] Web: www.cytekdev.com
Our mission is to offer complete solutions in the diagnosis of haematological diseases by flow cytometry. Our vision is to create a paradigm shift in the way of doing clinical diagnosis using flow cytometry.
Monday, 20 May
313
Cytognos S.L. is a biotechnology company based in Salamanca (Spain) dedicated to the design and development of new reagents, software and techniques that provide innovative solutions in the flow cytometry field.
Sunday, 19 May
Cytek Development, Inc.
Pol. La Serna, Nave 9, KM. 0 Santa Marta de Tormes Salamanca 37900 Spain Phone: 34623125067 Email:
[email protected] Web: www.cytognos.com
Saturday, 18 May
Coherent Inc. is one of the world’s leading suppliers of laser-based solutions offering reliability, cost, and performance advantages for the widest range of commercial and scientific research applications. Founded in 1966, Coherent designs, manufactures and markets laser systems and components, laser beam measurement and control equipment as well as leading-edge beam forming and beam guidance systems for manufacturers and scientific researchers across the globe.
Cytognos 721
Special Lectures
5100 Patrick Henry Drive Santa Clara, CA 95054 Phone: 408-764-4168 Email:
[email protected] Web: www.coherent.com
Congress Overview
Coherent, Inc.
81
Congress Overview
eBioscience, an Affymetrix company 723
Special Lectures
3420 Central Expressway Santa Clara, CA 95051 Phone: 408-731-5000 Email:
[email protected] Web:www.eBioscience.com
Sunday, 19 May
Saturday, 18 May
eBioscience, an Affymetrix company, develops and manufactures over 13,000 high-quality antibodies, recombinant proteins, immunoassays and multiplex assays at ISO-certified facilities worldwide for research and clinical use that accelerate scientific discovery in the areas of immunology and oncology.
EMD Millipore
814
Monday, 20 May
290 Concord Road Billerica, MA 01821 Phone: 781-533-6000 Fax: 978-715-1393 Email:
[email protected] Web: www.emdmillipore.com
Wednesday, 22 May
Tuesday, 21 May
EMD Millipore is the Life Science division of Merck KGaA of Germany, supporting research, development and production of biotech and pharmaceutical drug therapies. We support customers in cellular analysis, network elucidation and functional genomics with platforms for cell counting, simplified cell health assessment, benchtop flow cytometry and imaging flow cytometry.
Poster Session
Etaluma 222
Commercial Tutorials & Exhibits
1914 Palomar Oaks Way Suite 150 Carlsbad, CA 92008 Phone: 760-298-2355 Email:
[email protected] Web: www.etlauma.com
Speaker/Author Index
Poster Session Abstracts
Oral Session Abstracts
Etaluma Inc. is focused on bringing research-grade microscopy to a spectrum of users by decreasing the cost and complexity of these devices and encouraging the open-source development of application. Etaluma offers three models of inverted digital microscopy; the true color Model 400, the single 488nm fluorescence Model 500, and the new 3-color fluorescence Model 600. Etaluma is actively looking to provide hardware to reagent and kit manufacturers who want to expand their market into the economics of smaller labs.
82
EXBIO Praha a.s.
538
Nad Safinou II 341 Vestec 25242 Czech Republic Phone: 420 261 090 595 Email:
[email protected] Web: www.exbio.cz EXBIO Antibodies is a dynamic biotechnology company focused on design, development and manufacture of highest quality flow cytometry products (antibodies and kits) for IVD and RUO application, as well as on custom services at affordable prices. Besides world-wide distribution of its products under EXBIO label, the company sales many antibody reagents also on OEM basis. These products are widely recognized and appear in catalogues of large number of immunochemical retailers.
Flow Contract Site Laboratory, LLC 404 13029 NE 126th Place Kirkland, WA 98034 Phone: 425-821-3900 Fax: 425-821-3925 Email:
[email protected] Web: www.fcslaboratory.com Flow Contract Site Laboratory (FCS Lab) is a specialized Contract Research Organization (CRO) which offers expert services focused on GLP and non-GLP flow cytometry assays for research and development, nonclinical and clinical drug development. The management of our organization has resolved, as part of our diversification objective, to seek out partners who are interested in utilizing our capabilities to reduce existing work load while increasing flexibility and expanding their capabilities.
Fluidigm 631 7000 Shoreline Court Suite #100 South San Francisco, CA 94080 Phone: 650-266-6033 Email:
[email protected] Web: www.fluidigm.com Fluidigm develops, manufactures, and markets life science systems based on integrated fluidic circuits (IFCs). This technology furthers research by minimizing costs and enhancing sensitivity for applications such as single-cell gene expression, high-throughput SNP genotyping, and next-generation sequencing. Single-cell gene expression profiling has recently emerged as a powerful method to uncover
ISAC 2013 Program and Abstracts
808
440
840
11175 Flintkote Avenue San Diego, CA 92121 Phone: 858-642-0978 Fax: 858-642-0937 Email:
[email protected] Web: www.imgenex.com
Oral Session Abstracts
IMGENEX Corporation develops and commercializes reagents for human biology and disease for diagnostics or potential therapy. Products include a market leading portfolio of antibodies, reagents and LUCPorter™ Reporter Cell Lines for Innate Immunity, Tregs, Th17 cells, Dendritic Cells, and Inflammatory/ Immune Signaling Pathways for flow cytometry,
Poster Session Abstracts Speaker/Author Index
ISAC 2013 Program and Abstracts
IMGENEX Corporation
Commercial Tutorials & Exhibits
handyem develops and manufactures innovative flow cytometry solutions based on its patented, F3FiberFlowFluidics® microfluidics technology. handyem aims to democratize cytometry by providing day-to-day autonomy and agility to life science researchers with simple, highly efficient instruments at a fraction of the cost of today’s solutions. handyem’s HPC-100 Personal Cytometer, with its F3 microchip platform, brings simplicity, accessibility and ease of use to life science researchers.
iLab Solutions provides web-based research management services to scientific institutions. iLab’s research management and product selection tools enable researchers to share knowledge, track resources, manage budgets, and make smarter purchasing decisions. iLab also supports the work flow within core facilities to streamline request management, improve usage tracking and reduce non-compensation. iLab serves over 50 leading research institutions across North America and Europe.
Poster Session
360-2750 Einstein Street Quebec QC G1P 4R1 Canada Phone: 418-650-5999 Email:
[email protected] Web: www.handyem.com
Ten Post Office Square Boston MA, 02109 Phone: 617-297-2805 Email:
[email protected] Web: www.ilabsolutions.com
Wednesday, 22 May
handyem 719
713
Tuesday, 21 May
360 Foothill Road Bridgewater, NJ 08807 Phone: 908-231-0960 Fax: 908-231-1539 Email:
[email protected] Web: www.hamamatsu.com
iLab Solutions
Monday, 20 May
Hamamatsu Corporation
Hellma Analytics is the world’s leading manufacturer of custom flow channels for Cytometry and a reliable partner both for scientists and for instrument manufacturers. Sophisticated equipment and trained staff guarantees quality flow channels from prototyping to large scale production. Flow channel dimensions start as low as 50μm. State of the art features like cones to optimize hydrodynamic focusing, thin windows to fit large NA objectives, application of focusing lenses etc can be integrated at the highest precision.
Sunday, 19 May
GE Healthcare Life Sciences provides tools and technologies, solutions and expertise for drug discovery, biopharmaceutical manufacturing and cellular technologies that will enable research scientists worldwide to be more productive, effective, and creative. Our focus is to support you from idea to result.
80 Skyline Drive Plainview, NY 11803 Phone: 516-939-0888 Fax: 516-939-0555 Email:
[email protected] Web: www.hellmausa.com
Saturday, 18 May
800 Centennial Avenue, PO Box 1327 Piscataway, NJ 08855-1327 Phone: 732-457-8000 Fax: 877-295-8102 Email:
[email protected] Web: www.gelifesciences.com
520
Special Lectures
GE Healthcare
Hellma USA, Inc.
Congress Overview
heterogeneity in cell populations. In response to this, Fluidigm has developed a streamlined and automated workflow for capturing and analyzing single cells. The new C1™ Single-Cell AutoPrep System isolates single cells starting with low cell number input. For more information, visit our website at www.fluidigm.com
83
Congress Overview Special Lectures
immunofluorescent, or luminescent read-outs respectively. IHC-Pro™ antibodies are validated for immunohistochemistry and focused in Cancer and Immunopathology.
the ISAC booth to learn more about the society, meet society representatives such as the executive director or the editor-in-chief of Cytometry Part A, and discover how membership can help you advance your career.
Innova Biosciences
Life Technologies
502
Saturday, 18 May
Innova Biosciences, Babraham Hall, Babraham Cambridge CB22 3AT United Kingdom Phone: 044 1223 496 171 Email:
[email protected] Web: www.innovabiosciences.com
Tuesday, 21 May
Monday, 20 May
Sunday, 19 May
At the core of Innova’s business are LightningLink® and InnovaCoat®, innovative technologies that simplify antibody, protein and oligonucleotide labeling for use in R&D applications, drug discovery and the development of diagnostic kits. The easy to use, one step procedure allows scientists to label an antibody with 30 seconds hands-on time, saving both time and money. Antibody recovery is 100% and the technology is fully scalable without any deterioration in the performance of the labeled antibody.
IntelliCyt Corporation
907
Wednesday, 22 May
9620 San Mateo Boulevard NE Albuquerque NM 87113 Phone: 505-345-9075 Fax: 866-782-3140 Email:
[email protected] Web: www.intellicyt.com
Commercial Tutorials & Exhibits
Poster Session
IntelliCyt Corporation develops and markets screening solutions for Drug Discovery and Life Sciences Research. Our HTFC Screening System with new ForeCyt software provides superior speed, sensitivity and dynamic range for screening in 96 or 384 well formats. Our MultiCyt Screening Kits and robotic support combine to make this flow cytometry-based platform a complete solution for screening.
ISAC 742
Poster Session Abstracts
Oral Session Abstracts
9650 Rockville Pike Bethesda, MD 20814 Phone: 301-634-7454 Email:
[email protected] Web: www.isac-net.org
Speaker/Author Index
The International Society for Advancement of Cytometry (ISAC) is a scientific and educational organization that leads the way in development of cytometry, transfer of new methodologies, and exchange of cutting-edge scientific and technical information in quantitative cell sciences. Please visit
84
624
5791 Van Allen Way Carlsbad, CA 92008 Phone: 760-603-7200 Fax: 760-602-6500 Email:
[email protected] Web: www.invitrogen.com Life Technologies Corporation (NASDAQ: LIFE) is a global biotechnology company dedicated to moving science forward to improve life in meaningful ways for everyone. Our premier brands are the most cited, most trusted in the life sciences industry: Invitrogen™, Applied Biosystems®, Gibco®, Molecular Probes®, Novex®, TaqMan®, Ambion®, and Ion Torrent™.
Market Tech
213
1500 Green Hills Road, Suite 109 Scotts Valley, CA 95066 Phone: 831-461-1101 Fax: 831-461-1136 Email:
[email protected] Web: www.markettechinc.net Market Tech is a leading distribution and sales company offering a wide variety of Gas, Solid State, Diode and Fiber Lasers covering UV, visible and IR spectrum. We also offer laser power meters, light and color measurement instruments, optics and fiber optics products for life science, medical, aerospace, quality control and research applications.
Melles Griot
634
2051 Palomar Airport Road, 200 Carlsbad, CA 92011 Phone: 760-438-2131 Fax: 760-438-5208 Email:
[email protected] Web: www.cvimellesgriot.com Melles Griot designs and manufactures solid-state lasers, gas lasers and laser-based light engines. By working seamlessly with the customer from start to finish, we deliver high-value engineered solutions for Analytical Instrumentation, Bio-Instrumentation, and Ophthalmic and Medical Imaging applications. Melles Griot provides the perfect balance of performance, reliability, and manufacturability for OEM applications worldwide.
ISAC 2013 Program and Abstracts
512
University of New South Wales Sydney NSW 2052 Australia Phone: 612 9385 9813 Email:
[email protected] Web: www.nsinnovations.com.au/
Nexcelom Bioscience
8100 Southpark Way, A-8 Littleton, CO 80120 Phone: 303-730-1950 Fax: 303-730-1966 Email:
[email protected] Web: www.novusbio.com Novus Biologicals is a privately held, Coloradobased antibody and proteomics company. Novus provides the research community with over 186,000+ antibodies, proteins, peptides, RNAi, and research support products. Novus is a top tier proteomics company because of their high quality products, reliable customer service and technical support, as well as the Novus Guarantee+. Visit www.novusbio. com to learn more.
Poster Session Abstracts Speaker/Author Index
ISAC 2013 Program and Abstracts
804
Oral Session Abstracts
Newport, the world’s largest photonics company provides innovative solutions and industry leading product brands to multiple markets including the photonics and photovoltaic industries. Our combined broad product portfolio which includes: Corion®,ILX Lightwave™, New Focus™, Ophir, Oriel® Instruments, Richardson Gratings™, and SpectraPhysics® Lasers enables us to better partner with our customers to provide complete photonic solutions to make, manage and measure light.
Novus Biologicals
Commercial Tutorials & Exhibits
1791 Deere Avenue Irvine, CA 92606 Phone: 949-863-3144 Fax: 949-253-1680 Email:
[email protected] Web: www.newport.com
Nexcelom’s Cellometer line of simple-to-use cell counters automate manual cell counting procedures by obtaining accurate counts, viability, and cell sizes in less than 30 seconds & only 20uL of sample. Fluorescence detection capabilities enable fast & simple determination of GFP transfection rates, PIviability, & direct counting of WBCs without lysing.
Poster Session
504
360 Merrimack Street, Bldg. 9 Lawrence, MA 01843 Phone: 978-327-5340 Fax: 978-327-5341 Email:
[email protected] Web: www.nexcelom.com
Wednesday, 22 May
Newport Corporation
333
Tuesday, 21 May
Molecular Devices® is a leading provider of highperformance bioanalytical measurement systems for life science research, and biotherapeutic and pharmaceutical development. Our platforms for imaging cytometry, high content imaging, microscopy automation, image analysis, and microplate reader applications, enable scientists to improve productivity and effectiveness, accelerating research and discovery. Together through life sciences.
Monday, 20 May
1311 Orleans Drive Sunnyvale, CA 94089 Phone: 408-747-3545 Fax: 408-548-6431 Email:
[email protected] Web: www.moleculardevices.com
Sunday, 19 May
802
NewSouth Innovations are showcasing the outcome of research conducted in cluster identification for flow cytometry data. The software, named SOPHE, represents a breakthrough in processing flow cytometry data analysis. The SOPHE algorithms do not need any prior knowledge of the number of dimensions or clusters and it can find otherwise hidden relationships in multidimensional data. See us at booth 323; we would be happy to demonstrate the SOPHE system.
Saturday, 18 May
Miltenyi Biotec is a worldwide operating company offering comprehensive systems and services for life sciences, biomedical research and cellular therapies. The product portfolio includes state-ofthe-art instrumentation and reagents for cellular and molecular magnetic separation, cellular cultivation and flow cytometric analysis.
Molecular Devices, LLC
323
Special Lectures
Friedrich-Ebert-Strssa. 68 Bergisch-Gladbach 51429 Germany Phone: 49 2204 830630 Email:
[email protected] Web: www.miltenyibiotec.com
NewSouth Innovations Pty Ltd
Congress Overview
Miltenyi Biotec GmbH
85
Congress Overview
OSELA Inc.
605
Special Lectures
218 Brunswick Pointe-Claire QC H9R 1A6 Canada Phone: 514-426-2262 Email:
[email protected] Web: www.oselainc.com
Pavilion Integration Corporation
Sunday, 19 May
Saturday, 18 May
Osela is a specialized manufacturer of laser illumination systems for industrial applications in machine vision, life sciences and research. Our TOP HAT BEAM SHAPER efficiently transforms a laser beam (single mode and multimode) into at high uniform top hat profile with no high frequency noise. It’s compact, achromatic and easy to integrate into existing optical systems.
OXXIUS Lasers
506
Tuesday, 21 May
Monday, 20 May
4, rue Louis de Brogile Lannion 22300 France Phone: 33 6 74 15 09 18 Email:
[email protected] Web: www.oxxius.com
Wednesday, 22 May
Oxxius manufactures innovative solid-state lasers and systems for Life Science and Measurement applications. - LaserBoxx monolithic DPSS, available at 532, 553 and 561nm from 25 up to 300mW in very compact industry standard package (100x40x32mm).
Poster Session
- LaserBoxx Laser Diode modules at 405nm, 445nm, 473nm, 488nm, 515nm, 638nm. - LBX-4C Cost effective and modular 4 wavelengths combiner for laborary use.
Commercial Tutorials & Exhibits
Partec 830
Oral Session Abstracts
Am Flugplatz 13 Goerlitz, Saxony 02828 Germany Phone: 49 3581 87 46 0 Fax: 49 3581 87 46 70 Email:
[email protected] Web: www.partec.com
Speaker/Author Index
Poster Session Abstracts
Partec is a worldwide leading pioneer, developer and manufacturer of flow cytometry systems in a wide range of applications in healthcare, immunology, cell biology, microbiology, biotechnology, agrosciences, plant breeding, aquaculture and in pharmaceutical, food and beverage industries. Partec offers also innovative solutions for gel electrophoresis, PCR, fluorescence and transmitted light microscopy. With
86
own subsidiaries and specially trained distributors, Partec is active in more than 100 countries, worldwide.
729
2380-F Qume Drive San Jose, CA 95131 Phone: 408-453-8801 Fax: 408-453-8802 Email:
[email protected] Web: www.pavilionintegration.com
Phoenix Flow Systems, Inc.
630
6790 Top Gun Street, Suite 1 San Diego, CA 92121 Phone: 858-453-5095 Fax: 858-453-2117 Email:
[email protected] Web: www.phoenixflow.com Stop by to build up our egos by feigning interest in our products for: DNA analysis, MultiCycle, Apoptosis reagents, Apo-BrdU, Sheath concentrates, Cheap Sheath or Cell Detachment solutions, Accutase & Accumax while really only wanting to pick our brain about micro-brewery locations.
Photop Technologies Inc.
211
253 Fuxin East Road Fuzhou Fujian 350014 China Phone: 86 591 8805 2884 Email:
[email protected] Web: www.photoptech.com Photop Technologies, subsidiary of II-VI Corp. (NASDAQ:IIVI), is a leading photonics designer and integrated manufacturing company on Fiber Optics, Precision Optics, Projection and Display Optics, DPSS Laser, Crystal Materials, and other Photonics Products.
Propel Labs
623
131 E. Lincoln Avenue, Suite 200 Fort Collins, CO 80524 Phone: 970-295-4570 Fax: 970-372-5664 Email:
[email protected] Web: www.propel-labs.com Propel Labs, the developer of the Avalon, and now our partner Bio-Rad Laboratories, offers S3 the high performance bench-top cell sorter. The S3 is an easy to use 1-2 laser, 2-way sorter, with sort speeds >30k/
ISAC 2013 Program and Abstracts
Stratedigm, Inc.
522
19 Great Oaks Boulevard, Suite 10 San Jose, CA 95119 Phone: 408-884-4029 Fax: 408-351-7700 Email:
[email protected] Web: www.stratedigm.com
Commercial Tutorials & Exhibits
Stratedigm manufactures the SE520, S1000 and S1000Ex high-performance, bench-top flow cytometers upgradeable from one to four lasers with 14 Colors. Patented high-efficiency optics support user-configurable detector assignment and user-changeable filters for maximum performance. Our A600 High Throughput Auto Sampler offers a full automation solution compatible with all of Stratedigm’s analyzers. Our systems provide the best sensitivity, flexibility and reliability - truly, flow cytometers without compromise.
Oral Session Abstracts Poster Session Abstracts Speaker/Author Index
ISAC 2013 Program and Abstracts
Spherotech manufactures a variety of microparticles for flow cytometers. These particles are used for calibration, alignment, multiplexing, compensation, absolute counting and drop delay determination. Specifically, the calibration, alignment, and drop delay particles are used extensively for QC and long term performance tracking. In addition, Spherotech has particles for confocal fluorescence microscopy.
Poster Session
Sony Biotechnology Inc. is dedicated to helping the cytometry community of life scientists, researchers, laboratory professionals, & institutions achieve the best scientific results possible. We offer a comprehensive line of flow cytometry products, including our sy3200 & ec800 sorter/analyzer platforms & an extensive portfolio of fluorochrome conjugated antibodies & support reagents (>8000). We will also be showcasing Sony Corporation’s new advances in flow cytometry technology the SH800 and SP6800.
27845 Irma Lee Circle, Unit 101 Lake Forest, IL 60045 Phone: 847-680-8922 Fax: 847-680-8927 Email:
[email protected] Web: www.spherotech.com
Wednesday, 22 May
2100 South Oak Street Champaign, IL 61820 Phone: 217-979-1414 Email:
[email protected] Web: www.i-cyt.com
416
Tuesday, 21 May
303
Spherotech, Inc.
Monday, 20 May
Sony Biotechnology Inc.
Since 1982, SouthernBiotech has been dedicated to the production of the world’s highest quality secondary antibodies for research use. Now, we offer a broad range of polyclonal and monoclonal antibodies, including, fluorochrome and enzyme conjugates, Low Endotoxin/Azide Free preparations, F(ab’)2 fragments, purified immunoglobulins and collagens.
Sunday, 19 May
Semrock manufactures hard-coated optical filters that set the standard for biomedical & analytical instrumentation. Include the acclaimed VersaChrome® tunable bandpass filters, highperformance fluorescence & Raman spectroscopy filters & unique laser optics. Based on VersaChrome filters, Fresco™ tunable filter system provides a unique combination of high throughput & spectral flexibility for fluorescence microscopy & other spectral applications.
160 Oxmoor Boulevard Birmingham, AL 35209 Phone: 205-945-1774 Fax: 205-945-8768 Email:
[email protected] Web: www.southernbiotech.com
Saturday, 18 May
3625 Buffalo Road Rochester, NY 14624 Phone: 585-594-7000 Fax: 585-594-3898 Email:
[email protected] Web: www.semrock.com
SouthernBiotech 311
Special Lectures
Semrock 632
Congress Overview
sec, purity > 99%, a customized software platform and integrated sample cooling. Optional features for operator protection include a Class I and Class II Biosafety Cabinet. Propel Labs also offers MoFlo and CyAn upgrades including Co-Lase Towers and Nanoview for small particle detection.
87
Congress Overview
Thermo Fisher Scientific
633
Special Lectures
46360 Fremont Boulevard Fremont, CA 94538 Phone: 510-979-5000 Email:
[email protected] Web: www.thermofisher.com
Sunday, 19 May
Saturday, 18 May
Thermo Scientific Cyto-Cal and Cyto-Plex particles are designed to optimize flow cytometry performance. Cyto-Cal particles are ideal for calibration and setup, and for monitoring flow and optical stability. Cyto-Plex particles enable simultaneous detection and quantitation of multiple analytes within a single sample. Thermo Fisher Scientific is your single source for all your particle technology needs. Call our customer and technical support team at 1-800-2323342 and visit Booth #633 for details.
Monday, 20 May
Tonbo Biosciences
513
Tuesday, 21 May
4940 Carroll Canyon Road San Diego, CA 92121 Phone: 858-218-6626 Fax: 858-888-7301 Email:
[email protected] Web: www.tonbobio.com
Wednesday, 22 May
Tonbo Biosciences is a company built to serve your needs for flow cytometry reagents. Our goal is to do things differently, to fashion a better experience for you by providing the highest quality, high performance reagents at the best available prices.
Poster Session
Same Clones – established clones you trust and use every day Outstanding Quality – manufactured in the USA for the highest quality and performance
Commercial Tutorials & Exhibits
Exceptional Value – our products are priced at 30% to 50% less than your lowest priced supplier
Toptica Photonics Inc.
505
Oral Session Abstracts
1286 Blossom Drive Victor, NY 14564 Phone: 585-657-6663 Email:
[email protected] Web: www.toptica-usa.com
Speaker/Author Index
Poster Session Abstracts
TOPTICA is the world leader in diode laser and ultrafast technology for industrial and scientific markets. We offer the widest range of single mode tunable light in the 190 to 2900nm and 0.5-2.5THz spectral region with various accessories to measure, characterize, stabilize and analyze light. With our “Passion for Precision”, TOPTICA delivers!
88
Tree Star, Inc.
611
340 A Street, Building 1, Suite 203 Ashland, OR 97520 Phone: 800-366-6045 Fax: 541-482-3153 Email:
[email protected] Web: www.flowjo.com FlowJo is the next generation of flow cytometry analysis software. It handles your most ambitious projects with a high-level drag-and-drop user interface. Based on a patented experiment-based analysis paradigm, FlowJo intelligently handles protocols containing multiple tubes (any FCS files) with different samples stained with different reagent sets. Visit flowjo.com for the full story.
TTP Labtech Ltd
325
1 Kendall Square, Suite B2303 Cambridge, MA 02139 Phone: 617-494-9794 Fax: 617-494-9795 Email:
[email protected] Web: www.ttplabtech.com Would you like to multiplex your homogeneous beadbased assays for pennies per well? TTP Labtech is a global developer and manufacturer of high quality, innovative automated laboratory equipment for pharmaceutical and biotech research. Our portfolio of products minimize assay volumes, reduce material handling costs, and put the discovery tools back in the hands of the scientist. Visit booth #325 to find out more.
Union Biometrica, Inc.
711
84 October Hill Road Holliston, MA 01746 Phone: 508-893-3115 Email:
[email protected] Web: www.unionbio.com Union Biometrica Large Particle Flow Cytometers automate the analysis, sorting and dispensing of objects too big/fragile for traditional cytometers, e.g., large cells / cell clusters, cells in/on beads and small model organism. Choose between 4 COPAS models and the new BioSorter with interchangeable modules to cover the full 10-1500µm range.
ISAC 2013 Program and Abstracts
402
279 Campus Drive Stanford, CA 94305 Phone: 650-723-5054 Fax: 650-725-8564 Email:
[email protected] Web: www.woodsidelogic.com
516, 615
218 Xinghu Road C7-501 Suzhou Industrial Park Jiangsu 215123 China Phone: 86 051265928665 Email:
[email protected]
Tuesday, 21 May
We’re newcomers to flow cytometry instrumentation. We strive to make flow cytometers available to everyone who studies particles. Flow cytometers should be easy to use and affordable, but we don’t believe that compromising quality and performance will get us there. Because we are Xitogen Technologies - “excellence is in our genes.” Let’s work together. Come to Booth 615 and become one of the first to embrace a disruptive flow cytometer. Starting from $25k, upgradable to 4 lasers, 14FLs.
Wednesday, 22 May Poster Session
Vortran Laser Technology, Inc. is the newest and fastest growing provider of the highest quality laser solutions. Our products range from OEM laser modules up to fully integrated photonic sub-systems maximizing performance and profitability for our customers. The experience and capabilities of our engineering will expand your product performance while reducing costs allowing you to focus on the tasks you do best, not laser integration and optical alignment. Leave that to our design and expertise.
Xitogen Technologies Inc.
Monday, 20 May
21 Goldenland Court #200 Sacramento, CA 95834 Phone: 916-283-8208 Email:
[email protected] Web: www.vortranlaser.com
Sunday, 19 May
319
Integrated computer-based support for flow and other cell based studies: Use CytoGenie + AutoColor ? CytoHub ? EverTrieve ? AutoComp ? ClusterGenie ? DiffGenie ? CytoNote in Irisnote to plan analyses, collect, label and securely store data, do fluorescence compensation, sequentially choose axes to guide automated subset (cluster) identification, compare datasets, and integrate with other studies in a comprehensive well-indexed laboratory notebook cloud system.
Saturday, 18 May
Verity Software House, an industry leader in flow cytometry software development, offers a unique combination of innovative software for flow cytometry and unparalleled technical and customer support. ModFit LT, WinList, and GemStone: an unbeatable combination. Please stop by and see us in booth 402, and learn more about high-dimensional data analysis with GemStone at our Commercial Tutorial on Wednesday. Without Verity, it’s just software.
Vortran Laser Technology, Inc
938
Special Lectures
45A Augusta Road, PO Box 247 Topsham, ME 04086 Phone: 207-729-6767 Fax: 207-729-5443 Email:
[email protected] Web: www.vsh.com
Woodside Logic
Congress Overview
Verity Software House
Wiley 842 Commercial Tutorials & Exhibits
111 River Street Hoboken, NJ 07030 Phone: 201-748-5851 Email:
[email protected] Web: www.wileyblackwell.com
Oral Session Abstracts Speaker/Author Index
ISAC 2013 Program and Abstracts
Poster Session Abstracts
Wiley-Blackwell is the international scientific, technical, medical and scholarly publishing business of John Wiley & Sons, with strengths in every major academic and professional field and partnerships with many of the world’s leading societies. Wiley-Blackwell publishes over 1,400 peer-reviewed journals as well as 1,500+ new books annually in print and online, as well as databases, major reference works and laboratory protocols. For more information, please visit www.wileyblackwell.com.
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Congress Overview Special Lectures
Oral Abstracts 1
2
Apoptosis, Autophagy and DNA Damage
Cytometer Performance Characterization and Standardization
Saturday, 18 May
William Telford1, Zbigniew Darzynkiewicz2 1 National Cancer Institute, National Institutes of Health, Bethesda, MD, United States, 2Brander Cancer Research Institute, New York Medical College, Valhalla, NY, United States
Sunday, 19 May
Tutorial Objectives: In the first part of the Tutorial (Telford), flow cytometric methods to assess apoptosis will be thoroughly reviewed. Students will learn how to select the appropriate assay, the technical details necessary for assay success, and combining multiple assays for multiparametric analysis of cell. The tutorial will take a very practical approach, and actual cytometry data will be analyzed as part of the program. Autophagy, a phenomenon both related to and distinct from cell death will also be covered.
Monday, 20 May
In the second part of the Tutorial (Darzynkiewicz), detection of DNA damage by flow and image cytometry will be covered, both as a component of apoptosis and as a means of analyzing the effects of pharmacological, toxicological and environmental insults to live cells. Detection of both DNA damage itself and of phosphoproteins critical for DNA organization and structure that serve as markers for DNA damage will be particularly emphasized.
Wednesday, 22 May
Tuesday, 21 May
After completing the Tutorial, the student should be able to: (1) Have a working knowledge of flow cytometric assays for apoptosis, autophagy and DNA damage. (2) Be able to select an appropriate apoptosis and DNA damage assay for their experimental system, procure the necessary reagents and carry out the assay. (3) Combine multiple apoptosis assays to develop powerful systems for studying the complex signaling and progression of apoptosis.
Commercial Tutorials & Exhibits
Poster Session
Tutorial Details or Outline: Apoptosis and Autophagy (led by William Telford) 1) Types of cell death 2) Types of assays - DNA damage and loss (DNA binding dyes, TUNEL) - Cell membrane alterations and damage (DNA dyes, annexin V) - Caspase activation 3) Combining multiple apoptosis assays for more information 4) Choosing the right apoptosis assay 5) Critical parameters for analyzing apoptosis 6) Data analysis - artifacts and pitfalls 7) Analysis of autophagy by flow cytometry
Tutorial Objectives: Cytometers make optical measurements of particles. Using flow cytometers as the primary example, this tutorial will provide an understanding of the measurement process and how to critically and objectively characterize the instrument’s ability to make fluorescence and light scattering measurements. The use of standard instrument performance criteria and testing materials can allow minimal performance requirements to be established for an assay and assure that results on different instruments in different laboratories get comparable results when running the same sample. Authoritative standards (traceable to a standards setting organization) are generally not available for cytometry. Students will learn about the use and limitations of commercially available standard materials and some options for laboratory prepared standards. Tutorial Details or Outline: 1. Measurement characteristics that flow, scanning and static imaging cytometers have in common. 2. Characteristics of photomultiplier, solid state, and imaging detectors. 3. How to characterize critical instrument performance factors including: linearity, resolution, electronic noise, sensitivity (resolution at low signal levels), dynamic range. Reference methods and practical approaches to characterizing these factors are discussed. 4. How detailed characterization helps to troubleshoot problems. 5. Standardizing performance of one or more cytometers. 6. Status of authoritative calibration materials and practical alternatives. 7. Future possibilities for widely available standard performance criteria and materials for testing.
3
Approaches in Image-Based High Content Screening Susanne Heynen-Genel1, Jeffrey H. Price2,3 Conrad Prebys Center for Chemical Genomics, SanfordBurnham Medical Research Institute, La Jolla, CA, United States, 2NCI-Designated Cancer Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, United States, 3Vala Sciences Inc., San Diego, CA, United States
1
Tutorial Objectives: This tutorial will cover current approaches in image-based high content screening and provide examples. After attending this tutorial, the participant should have a good understanding of the currently available image-based screening methods and their advantages and limitations. Tutorial Details or Outline: 1. Basics of Image-Based High Content Screening 2. High-Throughput Imaging Assay Approaches 3. Advanced Methods for Image-Based Assays
Speaker/Author Index
Poster Session Abstracts
Oral Session Abstracts
DNA damage (led by Zbigniew Darzynkiewicz) 1) Detection and types of DNA damage in cells 2) Significance of DNA damage in both apoptotic and nonapoptotic cells 3) Role of pharmacological, toxicological and environmental insults in inducing DNA damage 4) Detection of histone-associated phosphoproteins as markers of DNA conformational changes and damage 5) Correlation of the above events with apoptosis and apoptosisassociated signalling events (i.e. caspase activity)
Robert Hoffman BD Biosciences, San Jose, CA, United States
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ISAC 2013 Program and Abstracts
6
Biosafety: Risk Assessment and SOP Development
Proliferation Tutorial: Cell Cycle Progression and Division – Unraveled by Flow Cytometry
High Throughput and High Content Screening for Flow Cytometry
Oral Session Abstracts
3. Cell Division We will discuss key assumptions involved using cell tracking dyes to monitor cell division based on decreasing dye intensity in successive daughter generations. Common problems encountered for each of the two major tracking dye types will be illustrated. • Covalent protein binding dyes (e.g. CFSE, CellTrace Violet, etc.) • Lipophilic membrane intercalating dyes (e.g. PKH26, CellVue Claret, etc.) • Applications of these methods to vaccine development, stem cell research and suppression assays
Poster Session Abstracts
4. Analysis of Cell Cycle Progression and Division Data The methods used to analyze cell cycle progression data obtained by flow cytometry will be discussed and demonstrated along with the methods used to quantitate cell division. To best understand
Speaker/Author Index
ISAC 2013 Program and Abstracts
2. Cell Cycle Progression This section will focus on the different methods for measuring cell cycle progression by flow cytometry. Labeling mechanisms and the information provided DNA binding dyes (“classic” cell cycle analysis) will be compared and contrasted with alternative methods including: • Thymidine Analogs (BrdU, EdU) • Proliferation-Related Antigens including cyclins and cyclin dependent kinases • FUCCI cell lines, vectors and mice
Commercial Tutorials & Exhibits
Tutorial Details or Outline: 1. Fundamentals of HT Flow 2. Assay Design for functional screening 3. Assays for Immunological screening 4. Automation and plate preparation 5. Running the assays 6. Data collection 7. Data analysis and processing 8. Reporting 9. Problems and solutions
1. Overview The DNA cell cycle will be reviewed providing everyone with a clear understanding of what needs to occur when in order for a cell to enter into and progress through the cell cycle before finally dividing. The differences between cell cycle progression and cell division and how flow cytometry is used to measure each will be explained.
Poster Session
After participating in this tutorial, the participant will be more aware of the advantages and possible disadvantages of having to work in an automated, or semi-automated régime. They will have a good knowledge of how to approach the preparation, and the analysis of data. They will also learn some analytical tools that have been designed particularly for high throughput and high content analysis.
Tutorial Details or Outline: Wednesday, 22 May
Tutorial Objectives: High throughput screening is now a real opportunity with flow cytometry. It is now possible to collect thousands of samples per day in a highly ordered way. However, designing and preparing these assays does require some careful attention to details and automated prep becomes an important component. One advantage is that you only sample very small volumes (like 1 uL) instead of 50 to 250 uL. This means instead of needing far more cells to start, you may actually be able to run far more samples than you might think. We will discuss several different implementations of HT flow cytometry and the types of assays that are usable in this format. This tutorial will also discuss aspects very high content flow cytometry where you might have a very large number of samples as well as a large number of parameters such as might be found in CyTOF data where parameters may exceed 20-30 per assay.
After participating in this tutorial attendees should have a clear understanding of 1) events that must occur in order for a cell to enter the cell cycle and to divide; 2) the difference between cell cycle progression and cell division as measures of proliferation; and 3) advantages and limitations of specific flow cytometric methods commonly used to quantify cell cycle progression and/or extent of cell division.
Tuesday, 21 May
J. Paul Robinson1, Padmakumar Narayanan2, Rob Jepras3 1 BMS/BME, Purdue University, West Lafayette, IN, United States, 2Amgen, Seattle, WA, United States, 3GSK, Middlesex, United Kingdom
Examples of applications using the different methods will be used to compare and contrast the information that is obtained using different probes and techniques. We will end with an opportunity for participants to discuss their real world problems and invite you to send your challenging cases to one of the instructors for discussion during the tutorial.
Monday, 20 May
5
Sunday, 19 May
Tutorial Details or Outline: Basic biosafety principles will be covered and in particular how the principles of containment and risk assessment apply to flow cytometry and cell sorting. The development of an SOP is a consequence of a thorough risk assessment and the steps required to formulate an instrument specific SOP will be presented. Procedures, engineering controls, disinfection, and personal protective equipment will be discussed as part of the overall SOP development process. An overview of the newly approved NIH Biosafety Policy for Cell Sorters will also be presented.
Tutorial Objectives: This tutorial will consider flow cytometric techniques for monitoring different aspects of cell proliferation. After a brief review of cell cycle biology, we will discuss: 1) Principles, methods and analysis strategies for measuring cell cycle progression using DNA binding dyes, thymidine analogues, proliferation-related antigens and phase specific expression vectors; 2) Principles, methods and analysis strategies for monitoring extent of cell division using proliferation tracking dyes (CFSE, PKH26 and newer analogs of each).
Saturday, 18 May
Tutorial Objectives: This tutorial will provide the attendee with an overview of biosafety principles, as they apply to flow cytometry with emphasis on cell sorting. The process of risk assessment for determination of biosafety containment levels and the development of a Standard Operating Procedure (SOP) for flow cytometers will be discussed.
Kylie Price1, Katharine A. Muirhead2, Paul K. Wallace3 1 Malaghan Institute of Medical Research, Wellington, New Zealand, 2SciGro, Inc./MidWest Office, Madison, WI, United States, 3Roswell Park Cancer Institute, Buffalo, NY, United States
Special Lectures
Kevin Holmes Flow Cytometry Section, NIAID, NIH, Bethesda, MD, United States
Congress Overview
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Congress Overview Sunday, 19 May
Saturday, 18 May
Special Lectures
these we will examine the evolution over time of these methods from the simple to the complex. • Cell Cycle Analysis Inside-out (S-Fit) Outside-in Non-linear least-squares analysis • Cell Cycle Progression Proliferative Fraction Proliferative Index Precursory Frequency
7
Tutorial Objectives:
Cell Sorting: Fundamentals, Applications and Troubleshooting
This tutorial will cover two subjects:
Monday, 20 May Tuesday, 21 May
Tutorial Details or Outline:
Wednesday, 22 May
The aim of this session is not to provide a "recipe book" type approach to electrostatic cell sorting, but rather to stimulate the participants to think about how cell sorting can be practically applied to help solve scientific problems.
Poster Session
The session will discuss how electrostatic cell sorting works and why it has become widespread, and compare this technology with other separation techniques. We will then cover the physical constraints that affect cell sorters and the implications they have on what goes into an instrument and what comes from an instrument. We will discuss ways that the cell sorter can make a "bad" decision and what to do about it.
Commercial Tutorials & Exhibits
8
Oral Session Abstracts
It is hoped that opportunities will arise for active audience discussion about cell sorting to occur.
Tutorial Objectives: Image-based experiments using cultured cells have proven to be a powerful means of generating informationrich data for biological applications. This tutorial will instruct biologists in the concepts and application of CellProfiler, an open-source, freely-downloadable software package designed for large-scale, automated phenotypic image analysis. We will work through hands-on examples, including construction of an analysis pipeline, optimization of module settings, automatic cellular feature detection and measurement, and exporting results. We will also briefly discuss the basic principles of supervised machine learning in order to score phenotypes where phenotypic differences between samples are subtle and/or complex. The tutorial participant should gain a working knowledge of CellProfiler and how to process and analyze their high- or low-throughput, high-content experiment.
Image Quantification and Analysis using CellProfiler David Logan Imaging Platform, Broad Institute of Harvard and MIT, Cambridge, MA, United States
Poster Session Abstracts
Analysis of Receptor Dynamics Using Flow Cytometry Alexandre Chigaev1, Yang Wu2 Pathology Dept. MSC09 5025, University of New Mexico Cancer Center, Albuquerque, NM, United States, 2Department of Pathology and Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, United States
Tutorial Objectives: 1. Provide information on the fundamentals of electrostatic cell sorting and an overview of the application of cell sorting in practical situations. 2. Discuss some of the areas where things can and do go wrong, and what steps can be taken to improve sorting experiments.
Speaker/Author Index
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5. Summary The tutorial will conclude with an open discussion using real world examples to illustrate what can and does go wrong and how best to avoid these problems.
Geoffrey Osborne Queensland Brain Institute / The Australian Institute for Bioengineering and Nanotechnology, Univ of Queensland, St Lucia, Brisbane, Australia
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Tutorial Details or Outline: 1. Basic overview of CellProfiler 2. Hands-on example(s) of an image-based assay analysis pipeline 3. Discuss other capabilities of CellProfiler, including interoperability with other tools 4. Example using a biologist-friendly machine learning tool, CellProfiler Analyst
1
Part I Real-Time Flow Cytometry of Integrin Signaling: Lessons Learned Have you ever dreamed of performing signaling experiments on live cells, in real-time, at natural receptor abundance, and without longterm pre-staining of cells using fluorescent dyes? We have been doing these for more than a decade. A unique property of integrin molecules is the ability to rapidly change ligand binding affinity, to “stand-up” on the cell surface, and to respond to the ligation of the binding pocket through the changes of the molecular conformation. These changes generate a plethora of molecular conformations, which, at the cellular level, are translated directly into different modes of cell-adhesive behavior. G-protein coupled receptors, tyrosine kinase receptors, and other signaling pathways (including nitric oxide/cAMP pathway) rapidly regulate integrin conformation, and modulate cell adhesion and mobilization, by triggering socalled “inside-out” signaling pathway. In the current presentation we focus on the basic methods and novel unpublished results including FRET-based measurement of molecular extension of LFA1 integrin, as well as rapid de-activation of VLA-4 integrin through previously undescribed signaling pathways. This work was supported by R01HL081062 and U54MH084960. Authors declare no competing interests. By the end of this section participants will not only learn about real-time flow cytometry, experimental design, and interpretation of the data, they will also understand the relationship between integrin molecular conformation and immune cell adhesive behavior (rolling, firm adhesion, or cell detachment). We will also discuss practical questions related to probe design, and commercial sources of existing probes. Part II High Throughput Flow Cytometry in Drug Discovery Now you’ve learned all the basics about monitoring surface protein signaling in real time, it’s time to move on to the hot field of drug discovery. In the second half of the tutorial, we will introduce you to a newly developed approach that is not only suitable for the real-time analysis of receptor trafficking, but also compatible with high-throughput flow cytometry (HTFC). The later application demonstrates that in addition to measuring individual samples from test tubes, flow cytometry has the capability to collect data from 40 samples per minute, and thus contribute to the early stage of drug discovery. In the sample approach, a newly available reporter protein (FAP) tag was fused with target protein, and ligand induced receptor trafficking was measured by flow cytometry in real-time. We will share with you some basics of HTFC and the FAP reporter system, experimental design of HTFC compatible assays, associated data analysis, and work flow. Videos taken in our high-throughput flow cytometry center at the University of New Mexico will also be available to demonstrate the automated processes of the sample plate preparation and screening.
ISAC 2013 Program and Abstracts
(Zucker an Fisher 2013, Protocols in Cytometry, Unit 1:28). This paper provides useful information in evaluating flow cytometers prior to purchase.
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Seeing a More Colorful World: A Guide to Polychromatic Flow Cytometry Pratip Chattopadhyay ImmunoTechnology Section, Vaccine Research Center, NIAID, NIH, Bethesda, MD, United States
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Analysis and Sorting of Rare Cell Populations
Robert Zucker1, Nancy Fisher2 1 Toxicology Assessment Division, U.S. Environmental Protection Agency, Research Triangle Park, NC, United States, 2 Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States
Tutorial Objectives: This tutorial will address how to find the proverbial ‘needle in the haystack’ when needing to identify or sort rare populations of cells. In the contemporary flow cytometry laboratory, there is often the need to accurate identify rare cells, such as circulating endothelial cell, endothelial progenitor cells, tumor cells, or immune subpopulations such as plasmacytoid or monocytoid dendritic cells.The student will gain an appreciation of obstacles in the accurate identification of rare cells and of strategies to overcome these obstacles and to assure better experimental data.
J. Philip McCoy NHLBI and CHI, Bethesda, MD, United States
Poster Session Commercial Tutorials & Exhibits
Tutorial Details or Outline: 1. Definitions, and an overview of the need for rare cell identification 2. Artifacts and sources of noise 3. Strategies for panel design and avoiding artifacts 4. Examples of rare event analyses 5. Verification of rare events, how do you know that you have identified the cells of interest with fidelity? 6. Summary and final thoughts
Oral Session Abstracts Poster Session Abstracts Speaker/Author Index
ISAC 2013 Program and Abstracts
Wednesday, 22 May
Tutorial Details or Outline: We will discuss instrument standardization, antibody conjugation and titration, panel development, troubleshooting, and data analysis.
Evaluation and Purchase of an Analytical Flow Cytometer: Some of the Numerous Factors to Consider
Tutorial Details or Outline: We discuss seven major issues to evaluate in the purchase of a new flow cytometer: • Applications, • Hardware and specifications (applications) ,
Tuesday, 21 May
Tutorial Objectives: This tutorial will cover the latest tips and tricks for successful polychromatic flow cytometry experiments. Participants will leave the session with a practical, working knowledge of how to develop or optimize the technology for their laboratories.
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Tutorial Objectives: 1) When purchasing a flow cytometer, the decision of which brand, model, specifications, and accessories may be challenging. The decisions should initially be guided by the specific applications intended for the instrument. However, many other factors need to be considered, which include hardware, software, quality assurance, support, service, and price and recommendations from colleagues. These issues are discussed to help guide the purchasing process. 2) Student should obtain information that will be used to evaluate different factors that are neccesary to evaluate in purchacing a new flow cytometer.
Monday, 20 May
In the first part of the tutorial, we will focus on a new approach to measure real-time receptor trafficking with high-throughput flow cytometry. We will be discussing: 1. Background and overview of high throughput flow cytometry and drug discovery 2. Methods for real time monitoring of receptor trafficking: design and development of HTFC compatible assays using FAP as the reporter protein tag. 3. De-convoluting the outcome from HTFC: primary screening, dose response screen and counter screens 4. Conclusions: New approaches to discover new ligands modulating receptor trafficking; Role of HTFC in drug discovery
Sunday, 19 May
Part II
Recommendations from colleagues. The individuals evaluating these machines should have an evaluation procedure in place to compare different units for their research endpoints. If the wrong unit is chosen, it may result in extra costs, lack of productivity, and less robust data that may not fulfill the current or future objectives of the laboratory. Our attempt is not to compare these machines or bead products used to test these machines directly, but to provide some insight on what factors to look for that may be overlooked by some scientists in their evaluation process. Instruments should be tested with the samples that are usually run in the laboratory. A series of quality assurance (QA) bead tests should be performed so resolution, sensitivity, and precision can be evaluated.
Saturday, 18 May
Part I In the first part of the tutorial, we will present the use of small ligand-mimicking fluorescent probes for the real-time analysis of receptor occupancy, affinity and conformational changes. We will discuss basics of real-time flow cytometry, as a novel approach that allows performing signaling experiments on live cells, in real-time, at natural receptor abundance. We will show how: • Real-time flow cytometry can be used for the determination of binding rate constants and dissociation constant • Rapid changes in the ligand binding affinity are detected under signaling through the cell receptors • Molecular extension (unbending) can be detected using FRETbased methods • Real-time binding of conformationally sensitive antibody is used for the determination of the dynamics of intracellular signaling
• Software, ease of use, and power, • Quality assurance testing, • Service, support, and company • Price and maintenance prices,
Special Lectures
Tutorial Details or Outline:
Congress Overview
By the end of the second half of this tutorial, we hope you will get some idea about the role of high-throughput screening in the field of drug discovery, gain some experience on setting up assays suitable for high-throughput flow cytometry, be familiar with a new type of biosensor, and bring home with some thoughts and ideas about how FAP and HTFC can do for your research.
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Congress Overview
13
Quantitative FRET Microscopy
Special Lectures
Gyorgy Vereb, Janos Szollosi Department of Biophysics and Cell Biology Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
Sunday, 19 May
Saturday, 18 May
Tutorial Objectives: The use of fluorescence (Förster) resonance energy transfer (FRET) for assessing molecular interactions in cellular systems is exponentially expanding. Several methods, mostly for microscopy, have been proposed, but most of them that made it to broad use owed to their simplicity suffer from being only qualitative or even from being prone to misinterpretation of results. The educational outcome of the tutorial is to provide the audience with stable foundations for applying a simple, yet qualitative FRET procedure that can be performed in any commonly available laser scanning fluorescence microscope. Addition outcomes include skills in interpretation of FRET data, and a broader knowledge on the pros and cons of various FRET methods that allow an educated choice of the approach most appropriate for the biological question.
Poster Session
Wednesday, 22 May
Tuesday, 21 May
Monday, 20 May
Tutorial Details or Outline: 1. Introduction: What is FRET? 2. Various manifestations of FRET: donor quenching and acceptor sensitization 3. The simplest generic approach to quantitate FRET from donor quenching: implementation, interpretation, limitations 4. The simplest generic approach to quantitate FRET from acceptor sensitization: implementation, interpretation, and the very serious limitations that make this approach the least recommendable (except a few specific cases) 5. Acceptor photobleaching (AccPb), as a self-controlled approach in measuring microscopic FRET from donor quenching - the method at its simplest 6. Artifacts that can arise during AccPb, and their correction 7. A simple ImageJ plugin for evaluating AccPb FRET 8. When time or space comes up as a 3rd dimension to be considered: basic principles of intensity-based (ratiometric) FRET measurements that are corrected for spectral bleedthrough 9. Summary, discussion, consultation for the participants about their ongoing experiments or plans.
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Commercial Tutorials & Exhibits
Growing a Cytometry Core Facility: Adding Calue with Hardware and Education Derek Davies1, Alfonso Blanco2 London Research Institute, Cancer Research UK, London, United Kingdom, 2Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland
Oral Session Abstracts
1
Speaker/Author Index
Poster Session Abstracts
Tutorial Objectives: Core facilities are now common in all work settings. Flow cytometry is a well-established technique but the core faces particular challenges in the face of expanding technology. In particular, cores need to bring added value to their users and institutional setting. But how can core facility staff keep up with the latest developments, how can they receive appropriate continuing education and how can this be passed on to users of a facility? We will discuss evaluation of technology and strategies for importing this into a core and also how education on site, at relevant meetings and by remote learning can benefit the facility. At the end of the tutorial the delegate will be aware of the approaches that can be taken to bring added value to the core, its staff and its users. Tutorial Details or Outline: This tutorial is aimed at Core managers or facility staff that work in established cores and who wish to
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expand the repertoire of service provision in times of budgetary restrictions and the ever-increasing time demands on a successful core. 1. Overview of the structure of a core facility: this includes facility planning, financial considerations, operational issues such as scheduling, assessment of users needs and re-charge. (10 minutes) 2. How is a facility judged? We will present how to assess the remit of the core and how to develop metrics that can be used to assess success and act as leverage to move the facility forward. (15 minutes) 3. How can the core grow? A successful facility needs to bring extra capacity or capability but how can new or alternative technologies be imported. What are the implications for the facility and its staff? (15 minutes) 4. New approaches to education. New hardware brings new challenges for education of facility staff. We will show how new approaches to training are required whether this is by internal training, external training, assisted sessions or remote learning. (30 minutes) 5. Collaborations between core facilities and other labs at national and international level to bring about standardisation. (15 minutes) 6. Summary and conclusions. (5 minutes)
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CellOrganizer: Building Models of Cell Structure from Microscope Images and Using Them for HighContent Screening and Cell Simulations Robert Murphy1,2, Gregory Johnson1, Devin Sullivan1 1 Lane Center for Computational Biology, Carnegie Mellon Univ, Pittsburgh, PA, United States, 2Department of Biological Sciences, Biomedical Engineering, Machine Learning, Carnegie Mellon University, Pittsburgh, PA, United States Tutorial Objectives: CellOrganizer is an open source software system that can learn models of the size, shape and spatial distribution of cellular components directly from images. These models are generative, which means that they can be used to synthesize new images of cells that are statistically similar to the ones they were trained on. Such images are useful for testing image analysis algorithms, and can be used as the basis for spatially-realistic cell simulations using systems such as Virtual Cell and MCell. Perhaps most importantly, CellOrganizer models represent a transportable means of representing the results of High Content Screening (HCS) assays that is not dependent on a specific instrument, assay or cell type. This tutorial will focus on how to use CellOrganizer and how to interface it with other software. The tutorial will begin with a brief overview of the conditional structure of the models within CellOrganizer and the system organization. The first part of the tutorial will focus on training generative models. Students are strongly encouraged (but not required) to bring a laptop. Attendees are also encouraged to bring a fluorescent cellular image dataset of their own to use for building a model, but datasets will be available at the tutorial for attendees who do not have one. Ideally, images should be two or three dimensional single cell images (i.e., already segmented) with different fluorescence channels for a fluorescently labeled target protein (ideally a protein showing a punctate or vescular pattern), a cell membrane or cytosolic-labeled marker, and a DNA marker (but these are not strict requirements). The second part will focus on synthesizing cell images from the models and importing the images or model parameters into other software systems. The last part will focus on adding new capabilities to the open source system, such as modules for building new types of components. Students should leave this session with mastery of the principles behind building probabilistic models from images and practical experience with training and using them with CellOrganizer. They
ISAC 2013 Program and Abstracts
forces present in the acoustic standing wave field to enrich bacteria in a continuous flow [7]. Further aspects on bacteria and acoustophoresis will also be discussed.
Tutorial Details or Outline: 1. Overview of conditional structure within CellOrganizer 2. Explanation of system architecture 3. Training 2D and 3D models for a. Nuclear shapes b. Cell shapes c. Vesicular or punctate proteins 4. Synthesizing in silico cells 5. Importing generated images into cell simulators 6. Using model parameters to analyze HCS experiments 7. Adding new functions
References
J. Dykes, A. Lenshof, I. Åstrand, T. Laurell, S. Scheding, PLOS One, August 2011, 6, 8, Brian Warner, Liping Yu, Marko Blom, Wilfred Buesink, Andreas Lenshof, and Thomas Laurell, CYTO 2012.
Persson J., Augustsson P., Laurell T. and Ohlin M., FEBS J, 2008, 275, 5657-5666
B. Björn Hammarström, T. Laurell, and J. Nilsson, Lab Chip, 2012, 12, 4296-4304 DOI: 10.1039/C2LC40697G
Thomas Laurell Measurement tech & Ind E E, Div. Nanobiotechnology, Lund University, Lund, Sweden
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The shape of cells is coupled to their environment by the actions of signaling networks that dynamically regulate complex interactions amongst components of the cytoskeleton, membrane, and cellsubstrate adhesions. But very little is understood as to how signal transduction results in specific cellular forms. In my laboratory we are trying to answer three broad questions: 1) How does signaling network activity generate shapes that are important for behaviors such as cell migration? 2) How do networks “sense” cellular shapes? 3) When the systems that regulate cell shape fail, or are re-engineered (e.g. cancer), what are the consequences? In order to answer these questions we continue to assemble, using high-throughput and high-content imaging, a compendium of data describing the shape of millions of single cells from different genetic backgrounds, across different growth conditions, and following chemical perturbation or RNAi. We then implement novel computational methods to analyze this “data cube”, and model signaling networks that control cell shape.
Wednesday, 22 May Poster Session
Here I will discuss how we have gained new insights into how signaling network activity underpins the morphological heterogeneity of populations, and how the activity of key transcription factors such as Nf-kappaB and YAP/TAZ activity is influenced by this heterogeneity. Firstly, we have performed genome-scale RNAi screens in Drosophila and found that wildtype Drosophila hemocytes and neuronal cells are remarkably heterogeneous, and exist in a number of discrete stable states. We show how signaling activity that drives transitions between stable shapes, and that morphological heterogeneity is a structured, tunable, and evolvable process. We also validate our findings made in Drosophila using models of metastatic melanoma. Secondly, through analysis of a chemical screen in breast cancer cells, we quantify how Nf-kappaB and YAP/TAZ nuclear translocation is not only dependent of the shape of individual cells, but also on the shape of neighboring cells. We provide both systems-level and mechanistic insights into how the shape of cells can modulate transcriptional activity. These findings have consequences as to how morphological heterogeneity can influence transcriptional programs and ultimately disease progression.
Commercial Tutorials & Exhibits Oral Session Abstracts Poster Session Abstracts Speaker/Author Index
ISAC 2013 Program and Abstracts
Chris Bakal Cancer Biology, Institute of Cancer Research, London, United Kingdom
Tuesday, 21 May
A most challenging area for acoustophoresis is the processing of samples with particles smaller than 1-2 micrometers since the primary acoustic radiation force scales to the third power of the radius. This also explains why there are only a few reports on acoustophoresis for bacteria handling. Recent findings have however opened up the possibility of using the secondary acoustic
Signaling Networks Regulating Cellular Growth and Form
Monday, 20 May
Acoustophoresis intrinsically offers simple means for continuous flow based sample preprocessing and clean-up prior to analysis. Examples will be given where cell debris and dissolved labels are removed prior to FACS redout using acoustophoresis [3]. On-line sample preparation of dairy sample for somatic cell counting has also been reported where interfering lipid particles are removed prior to either Coulter Counter analysis of fluorescence readout facilitating the endpoint analytical step [4]. Using affinity ligands coupled to microbeads acoustophoresis further enables targeted extraction of molecular and cellular species. Examples are given where allergy antigen specific bacteriophages are extracted from a phage display library [5] and molecular species are enrichment and purifiedin an acoustophoretic sample pre-processing step before mass spectrometry readout [6].
Sunday, 19 May
Translating Acoustophoretic Cell Handling to Clinical Applications
FFA has also been developed to deplete thrombocytes in acentrifugation-based peripheral blood progenitor cell (PBPC) apheresis, intended for stem cell extraction inclinical therapy of haematological disorders [2].
Saturday, 18 May
Carl Grenvall, Jacob Riis Folkenberg, Per Augustsson, Thomas Laurell, Cytometry A, 2012, 81A, 12, 1076-1083
P. Augustsson, J. Persson, S. Ekström, M. Ohlin and T. Laurell;Lab Chip, 2009, 9, 810–818
Free Flow Acoustophoresis, FFA, utilizes the fact that individual cell types display cell different migration velocities in an acoustic standing wave field, which enables continuous flow based separation. More recently FFA has been developed to target clinical use where purification of tumor cells (TC) from white blood cell fractions have been accomplished [1]. A key question in this respect is whether FFA, which is a labelfree separation, can provide TC populations that are not detected (i.e. not expressing EpCAM) by means of the current clinical standard method –Veridex.
Special Lectures
Per Augustsson, Cecilia Magnusson, Maria Nordin, Hans Lilja, Thomas Laurell, Anal Chem, 2012, 84, 7954-7962
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Acoustophoresis in microfludic systems is gaining attention as a viable technology platform for continuous flow based cell manipulation, including cell separation, buffer exchange, valving, concentrations, affinity extraction and cell interaction studies. We have recently shown (submitted) that cells processed by means of microchip acoustophoresis experience a low mechanical stress and display no change in biological response when compared to control samples. Thisnow opens the route to the development of a wide range of clinical applications where several of the unitoperations in standard cell assay protocols can be replaced by acoustophoretic cell processing.
Congress Overview
will be able to use them to compare results from different HCS assays using the generative model parameters, and import synthetic images into cell simulation systems.
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Congress Overview
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Discovery of Small Molecules that Control Cell Differentiation
Special Lectures
Petr Bartunek Institute of Molecular Genetics, Prague, Czech Republic
20 Saturday, 18 May
Expanding the Capabilities of Mass Cytometry Scott Tanner, Alexander Loboda, Dmitry Bandura, Vladimir Baranov, Olga Ornatsky DVS Sciences Inc., Markham, ON, Canada
Sunday, 19 May
The Mass Cytometer is a specific, designed-for-purpose implementation of an analytical atomic mass spectrometer. The principal point of commonality is the Inductively Coupled Plasma that provides atomization and ionization. This presentation will focus on the unique adaptations that address the need for single cell distinction, with the sensitivity and dynamic range that meet the needs of biologic informatics.
Wednesday, 22 May
Tuesday, 21 May
Monday, 20 May
The overall efficiency of a Mass Cytometer system in quantifying antigens in real time single cell assays depends on several factors. First, processes of aerosol generation, vaporization, atomization and ionization of single cells define how quantitatively the singlecell induced ion cloud sampled through the plasma-vacuum interface represents the composition of each single cell. Second, ion transport through the interface, the ion optical path and the time-of-flight analyzer define not only the absolute sensitivity (effectively defining the minimum number of copies of antigen per cell that can be detected given a specific cell-labeling efficiency), but also how readily bright and dim labels can be distinguished while retaining the specificity and high multiparameter capability of the assay. Third, ion signal handling on the nano-second scale, which includes ion detection, signal pre-amplification, digitization and processing, defines the dynamic range of a single cell assay. Integration of the ion signals over the mass-time windows, combined with appropriate real-time Gaussian curve fitting of the sequential spectra corresponding to microsecond scale transients, provides additional gating information to resolve doublets and debris from true single cell events.
Poster Session
Using leukemia cell lines, we will demonstrate how the theoretical fundamentals of these sequential processes can be translated into hardware and software improvement of data fidelity.
21 Commercial Tutorials & Exhibits
Simultaneous Analysis of Multiple Fluorescent Proteins and Fluorochromes by a Novel Spectral Flow Cytometer
Oral Session Abstracts
Michio Tomura1, Koji Futamura2, Nao Nitta2, Masaya Kakuta2, Motohiro Furuki2 1 Center for Innovation in Immunoregulative Technology and Therapeutics, Kyoto University Graduate School of Medicine, Kyoto, Japan, 2Bio Science Business Department, Life Science Business Division, Medical Business Unit, SONY Corporation, Tokyo, Japan
Speaker/Author Index
Poster Session Abstracts
Introduction: We have already presented various biomedical applications using spectral flow cytometry (FCM) at CYTO1-4. In this presentation, we show the simultaneous analysis of multiple fluorescent proteins (FPs) and fluorochromes with spectral unmixing that improve signal resolution and reproducibility over traditional filter based cytometers. FPs are a powerful reporter system in biomedical research fields. The ability to detect multiple FPs and fluorochromes simultaneously using FCM provides the opportunity to differentiate among various cell populations, or to study gene function and monitor protein-protein interactions in individual
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cells. However, traditional flow cytometric analysis of multiple FPs and fluorochromes has been difficult. We show several results of simultaneous analysis including photoconvertible FPs such as Kaede and KikGR by spectral FCM with a unique algorithm5. Methods: We have further developed a novel spectral FCM as previously reported3. Unlike traditional FCMs, our novel spectral FCM uses a full spectrum 32 channel linear array PMT to detect the entire fluorescence derived from all fluorescent probes over a range from 500 to 800 nm. These spectra are analyzed to automatically deconvolve the contribution of every fluorochrome, even in the presence of significant spectral overlap, enabling more flexible experimental design. One of the advantages of spectral FCM is the recognition of the unique spectral emission profiles of each fluorochrome, and is not limited to merely the peak wavelength. To further exploit this strength, we analyzed various cells labeled with adjacent FPs and fluorochromes such as GFP/FITC, KikGR-Green/ FITC, GFP/Venus (YFP variant), PE/KikGR-Red, and Fucci/KikGR, where the emission peaks have almost identical wavelengths, but the fluorochrome spectra differ. We have successfully demonstrated eleven color analysis including KikGR-Green and -Red simultaneously with bright and very useful fluorochromes such as PE, PE-Cy5, and APC of which the combination is indistinguishable on a traditional cytometer. We also analyzed and compared spectral changes of KikGR during photoconversion by both spectral FCM and confocal microscope. Results: Using spectral FCM, we were able to demonstrate successful deconvolution and identification of respective FP signals. We also analyzed KikGR during photoconversion and detected the increase of KikGR red signal together with the concomitant decrease of KikGR-Green spectra in a time dependent manner. There were good correlations between spectral changes by spectral FCM and color images obtained by confocal microscopy. Examining EGFP and EYFP labeled cells, the spectral FCM was able to clearly distinguish EGFP+, Venus+ and EGFP+Venus+ populations. These data show that spectral FCM has a reliable deconvolution of multiple, spectrally overlapping fluorochromes enabling the simultaneous and reliable detection of multiple FPs despite varying spectral overlap or dramatic differences in relative intensities. Conclusion: The improved spectral FCM provides the most reliable and accurate analysis of multiple FPs and fluorochromes. Our presentation will include updates on our spectral flow system and application results at the conference in May 2013. References: Nobukazu Watanabe, CYTO2011 2 William Hyun, CYTO2011 3 Koji Futamura, CYTO2011 4 Nao Nitta, CYTO2012 5 Masashi Sekino, CYTO2012 1
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Quantitative Real Time Single Cell Spectroscopy in Flow John Nolan1, Danilo Condello, Erika Duggan2 1 La Jolla Bioengineering Institute, La Jolla, CA, United States, 2 La Jolla Bioengineering Institute, San Diego, CA, United States Introduction: The long standing interest in measuring the complete emission spectra from individual cells in flow cytometry has recently become a practical reality. Advances in optics, detectors and electronics have enabled several approaches for single cell spectroscopy in flow, opening the door to new instrument concepts, labeling strategies, and biological applications. Key to continued progress in this area is a systematic evaluation of calibration, standardization, and data analysis approaches. We have developed high resolution spectral flow cytometers capable of high speed fluorescence and Raman scattering measurements. Here we report on the sensitivity and dynamic range of these spectral flow
ISAC 2013 Program and Abstracts
Conclusions: This new measurement system preserves the efficiency of traditional time-domain systems as well as throughput of standard cytometry. It does not require frequency modulation that traditional phase sensitive instruments require. In the future we plan to evaluate the instrument for multiple-exponential fluorescence decays, for exploring the fluorescence lifetime of various locations across a cell, and for implementing new biological applications.
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Poster Session Abstracts Speaker/Author Index
ISAC 2013 Program and Abstracts
Oral Session Abstracts
Conclusion: We report remarkably durable Ag85A-specific CD4 T cell responses up to 6 years after MVA85A vaccination. We propose
Commercial Tutorials & Exhibits
Methods: A flow cytometer was developed based on a traditional design but incorporating a rapidly scanning cw laser beam
Poster Session
Results: We successfully completed long-term follow-up in ~75% of participants previously vaccinated with MVA85A. The maximum follow-up time, in adults, was 6 years after MVA85A vaccination, while the minimum follow-up time was 3 years, in infants. Frequencies of Ag85A-specific CD4 T cells were highly durable in all ages: response magnitudes significantly exceeded the prevaccination responses. Magnitudes of Ag85A-specific CD4 T cells in MVA85A recipients were also significantly higher than those of placebo recipients.These CD4 T cells almost exclusively expressed Th1 cytokines and were predominantly polyfunctional (coexpressed IFN-g, TNF-a and IL2).These antigen-specific Th1 cells displayed CCR7-CD45RA- effector memory or CCR7+CD45RAcentral memoryphenotypes, in roughly equal proportions.
Wednesday, 22 May
Background: Mycobacterium tuberculosis (Mtb) is one of the most pervasive diseases today. The aim of prophylactic vaccination is to induce long-lived immunity that provides a rapid recall response upon pathogen encounter. Heterologous prime-boost regimens may constitute the most promising vaccination strategy against tuberculosis (TB). MVA85A is a new TB vaccine, which is designed to boost immunity primed by the current TB vaccine, BCG. However, whether such prime-boost strategies induce long-lived antigen-specific memory responses is not known. The aim of this study was to determine if specific T cell responses persist up to 6 years after vaccination with candidate heterologous boost vaccine, MVA85A.
Background: Fluorescence decay measurements using a timedomain approach is a powerful and sensitive technology when combined with epifluorescence microscopy. Time domain measurement systems observe the fluorescence decay of molecules, metabolites, or other fluorescent species inside of cells. When images are acquired by fluorescence lifetime imaging microscopy (FLIM) or similar relatives, highly resolved multi-pixel data can be obtained. Lifetime values help indicate concentration independent subcellular phenomena. Although FLIM systems retain high signal to noise, they are still limited in efficiency and throughput. In this contribution we present a new type of flow cytometer designed to rapidly capture fluorescence decay profiles based on a time-domain approach.
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Tuesday, 21 May
Erica Smit1, Michele Tameris1, Elisabeth Jane Hughes1, Ashley Veldsman1, Linda van der Merwe1, Hennie Geldenhuys1, Mark Hatherill1, Helen McShane2, Willem Hanekom1, Hassan Mahomed1, Thomas Scriba1 1 University of Cape Town, Cape Town, South Africa, 2University of Oxford, Oxford, United Kingdom
Wenyan Li1, Giacomo Vacca2, Mark Naivar3, Jessica Houston4 New Mexico State University, Las Cruces, NM, United States, 2 Kinetic River Corp., San Jose, CA, United States, 3DarklingX, LLC, Los Alamos, NM, United States, 4Chemical Engineering, New Mexico State University, Las Cruces, NM, United States
Fluorescence Lifetime-Dependent Flow Cytometry in the Time-Domain
Monday, 20 May
Remarkable Durability of Ag85a-Specific CD4 T Cell Memory Responses up to 6 Years after Mva85a Vaccination
Methods: We recalled adults, adolescents, children and infants, who previously received a single intradermal MVA85A vaccination. Peripheral blood mononuclear cells (PBMCs), were isolated and frequencies of Ag85A-specific T cells measured by IFN-g ELISpot assay. We characterized the T cell response in more detail with a whole blood intracellular cytokine staining (ICS) assay. Intracellular expression of IFN-g, TNF-a, IL-2 and IL-17 by CD4 and CD8 T cells, as well as memory phenotypes of these cells (using CCR7 and CD45RA), were measured by multiparameter flow cytometry, using a nine-color antibody panel.
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Sunday, 19 May
Conclusions: Spectral flow cytometry can offer the sensitivity and dynamic range of conventional flow cytometry, with the added benefits conferred by measurement of complete spectra from individual cells. These benefits include: a straightforward approach to report results quantitatively, improved resolution of overlapping spectra, and the possibility, with high resolution systems, to implement new types of labels such as nanoparticle SERS tags. Supported by NIH EB003824.
Results: In our initial evaluation we measured fluorophores with different lifetime values ranging approximately from 3 ns to 25 ns. Lifetime decay profiles from microspheres and cells showed differently sloping fluorescence decays, in comparison to scatteronly pulses.
Saturday, 18 May
Results: Calibration of the spectral flow cytometers with fluorescence intensity standards enabled us to characterize detection efficiency (Q, photoelectrons per MESF). The sensitivity and dynamic range of the spectral flow cytometers were at least as good as our benchtop conventional flow cytometer. We measured the binding capacity of antibody capture beads, and used these calibrated capture beads to acquire reference spectra from fluorescence- or SERS-labeled antibodies. These reference spectra confer information about the spectra of individual antibodies, as well as the brightness, enabling the spectral analysis to report either the brightness of a cell (in units of photons or MESF) or the abundance of a label in/on a cell (in units of antibodies per cell). A comparison of real-time spectral unmixing with post-acquisition spectral analysis off line gave equivalent results in terms of per cell tag abundances, and paves the way for the development of spectral cell sorters.
excitation source. The laser was focused to a tight spot size to yield short interaction pulses when scanned over cells or microspheres between 2 and 10 microns in diameter. The fluorescence decay was deconvolved from the raw fluorescence and scattering cytometric waveforms, which were collected using a high-speed data acquisition system.
Special Lectures
Methods: Our spectral flow cytometers employ imaging spectrographs with holographic gratings to disperse the light across high speed CCD detectors for high resolution spectral measurements, and mirrors, filters, and PMTs/PDs for conventional measurement of discrete spectral bands. The data acquisition system provides full control of all detectors, as well as real time display and spectral analysis of individual cells. Beads and cells were stained with fluorescent antibodies and/or antibodies labeled with surface enhanced Raman scattering (SERS) nanoparticle tags. The spectral data were analyzed either by integrating the intensity across specific spectral ranges, creating the equivalent of virtual bandpass filters, or by classical least squares (CLS)-based spectral unmixing of the spectra using the known spectra of labels, autofluorescence and background signals as reference components. We used calibrated intensity standards as well as reagent-capture beads to calibrate the brightness and target abundance on unknown samples.
Congress Overview
cytometers, and illustrate how spectral overlap and quantification of both cell brightness and target abundance can be addressed using spectral unmixing.
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Congress Overview
that suchlong-lived immunity represents a highly desirable attribute of the T cell response induced by heterologous prime-boost regimens against TB.
25 Special Lectures
Functional Characterization of Lymphoid Subsets in Chronic Myelogenous Leukemia by Mass Cytometry Phospho-Flow Analysis
Saturday, 18 May
Jitakshi De1, Rosemary Fernandez 1, Neil Shah2, Holden Maecker1 1 Human Immune Monitoring Center, Stanford University, Stanford, CA, United States, 2UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, CA, United States
Monday, 20 May
Sunday, 19 May
Background: Mass cytometry (MC) is a novel approach to precisely decipher cell-specific activity of cytokine-mediated signaling in disease states, allowing simultaneous assessment of different celltypes in a complex biologic milieu. Despite enormous success of tyrosine kinase inhibition (TKI) in chronic myelogenous leukemia (CML) therapy, there is a 20-30% rate of relapse. TKI-refractory CML stem/progenitor cells survive independent of BCR-ABL kinase activity. Identification of residual CML cells and characterization of mechanisms underlying therapy resistance and relapse are necessary for cure. Here, we applied MC phospho-flow to study potentiated oncogenic pathways in chronic phase and relapsed CML.
Wednesday, 22 May
Tuesday, 21 May
Methods: Phospho-flow analysis was performed on fresh blood samples from two patients with CML (one treatment-naive in chronic phase, and the other with prior TKI therapy and a rising BCR-ABL1 transcript) referred to UCSF Comprehensive Cancer Center, in parallel with a healthy donor sample. Aliquots of each were treated with IL3, IL6, or no stimulus, for 10 min at 37C, lysed and fixed, and reacted with a series of rare earth metal isotopetagged antibodies including those towards lineage-determining, activation, and maturation antigens; phosphorylated epitopes within key regulatory proteins of JAK/STAT and MAPK pathways; and IKB kinase. ICP-MS data acquired on CyTOFTM mass cytometer at Stanford HIMC were analyzed on high-dimensional data analysis algorithms.
Poster Session Abstracts
Oral Session Abstracts
Commercial Tutorials & Exhibits
Poster Session
Results: 1. In chronic phase CML, subsets of CD19+ lymphoid cells had differential STAT5 activation profile: IgD+/CD27-/CD33- naive B cells had no baseline p-STAT5, but had marked (~29.2X) IL3induction; whereas IgD+/CD27+/CD33+/IL3R+ cells had high baseline p-STAT5 and diminished (~2.5X) IL3-induction. IgD-/ CD33-/IL3R-/CD45dim progenitors had low baseline, and no inducible STAT5 activity. 2. Compared to cellular counterparts from the healthy donor, baseline p-STAT3 in CML cells was much lower with substantial IL3 and IL6 induction. While the naive T cell compartment was largely preserved in chronic phase, it was diminished in relapsed CML. 3. In the relapsed case, rare IL7R+ subpopulations (CD19br/IgD-/ CD45dim progenitors and CD27+/CD45RA+ naive Th cells, both comprising 250,000 full wells/day (4.3min per 384- or 8.5min per 1536-well plate) a reality. Performing autofocus in parallel with continuous motion imaging allows for a much larger percentage of the overall scanning time to be devoted to light collection, thereby removing many of the inefficiencies inherent in currently available instruments and making image brightness and signal-to-noise a function of light source and assay brightness.
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Automated Intracellular Fret Measurements using Hyperspectral Microscopy and Feature Extraction Silas Leavesley1,2, Andrea Britain3, Thomas Rich2,3 Chemical and Biomolecular Engineering, University of South Alabama, Mobile, AL, United States, 2Center for Lung Biology, University of South Alabama, Mobile, AL, United States, 3Pharmacology, University of South Alabama, Mobile, AL, United States 1
Background: Förster resonance energy transfer (FRET) is a fluorescence microscopy tool that has been invaluable in
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understanding spatially-dependent phenomena in living cells. Many techniques have been implemented to quantify the level (or efficiency) of energy transfer. These range from simple estimates based upon the intensity of one or two detection bands to complex experimental approaches and equipment configurations, such as fluorescence lifetime or acceptor photobleaching. These techniques have advantages and disadvantages, which have been widely documented and debated. For example, fluorescence lifetime calculations can be made insensitive to changes in total fluorophore concentration, which is advantageous when samples have varying fluorophore concentrations. However, identification of many fluorescent signals, in addition to the FRET pair, is very difficult to perform using lifetime and photobleaching techniques. Hyperspectral microscopy techniques may also be used to measure FRET efficiency.In theory, hyperspectral microscopy and spectral flow cytometryapproachesallow measurement of FRET efficiencies in experiments with many fluorescent labels and high tissue autofluorescence. Methods: In this work, we have combined hyperspectral microscopy with automated image analysis and feature extraction to measure subcellular FRET efficiencies in time-lapse confocal microscopy studies. HEK-293 and pulmonary aortic endothelial cells were grown on 25 mm round coverslips and transfected with either a cytosolic or plasma membrane-localized CFP-EPAC-YFP probe. EPAC changes conformation upon binding cAMP; the CFPEPAC-YFP probe displays approximately 45% FRET efficiency at basal cAMP levels and 37% FRET efficiency at saturating cAMP. Two days post-transfection, cells were labeled with Hoechst 33342 and imaged on a Nikon A1R spectral confocal microscope (405 nm excitation, 432-606 nm emission in 6 nm increments). Results: Non-negatively constrained linear unmixing was used to calculate the abundance of Hoechst, CFP, and YFP. Image processing and feature extraction using Cell Profiler (The Broad Institute) software was then performed. Time-lapse single-cell FRET efficiencies (whole cell and cytosolic) were then calculated. Initial tests with forskolin and rolipram displayed a characteristic increase in cytosolic cAMP with an average standard error-of-the-mean of 0.9% (over the time-course, n=8 trials). Conclusions: These results indicate that hyperspectral imaging or flow cytometryapproaches can be used to measure FRET efficiencies in the presence of additional fluorescent labels – and potentially autofluorescence – with high accuracy and precision. In image cytometry experiments, additional labels would yield valuable information for feature extraction, such as nucleus identification, as shown here. Future work will focus on localizing FRET signals to specific subcellular domains and performing studies in whole-vessel pulmonary vascular preparations.
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Amplifying DNA Beads Assay in Flow Cytometry: A Platform Technology Based on Exonuclease III-Aided Target Recycling Jie Lu, Dayong Jin Macquarie University, Sydney, Australia Introduction: Exonuclease III(Exo III) has been recently discovered to “recycle” target molecules, thus resulting in the PCR-like sensitivity1,2. Here we reportapplication of Exo III amplification technique to flow cytometry, yielding a highlysensitive, reproducible, separation-free, and high-throughput DNA quantification platform. Method: The schematic diagram was shown in Fig. 1. TheExo III can catalyse the stepwise removal of mononucleotides in a 3’ to 5’ direction from probe’s 3’ blunt end, removing fluorescence reporter and ultimately releasing the target. The released target can then recycle to hybridize withsecondarysurface DNAprobes in sequence, thereby improving sensitivity. Owing to the high-density coverage of carboxyl groups on polystyrene beads, different ratios of Cy5
ISAC 2013 Program and Abstracts
Methods: We are developing an innovative approach (GlycoSenseTM) capable of real-time glycoprofiling based on combining multiplex suspension array technology with glycanspecific reagents. In this method, flow cytometry is used to detect binding between glycans and glycan-specific reagents that are conjugated to spectrally-unique microspheres. By combining the individual reagents into a multiplex suspension array, the entire analysis can be obtained in less than a minute on a basic cytometer.
Saturday, 18 May
Conclusion: This work shows the great potential to apply Exo III recycling technique to flow cytometry, leading to both high sensitivity and analysis speed.
lead times also make corrective action during commercial cell cultivation impossible, with the only recourse to specification failures being lot rejection, at a cost of $5-$50M. The availability of robust, rapid, and simple analytical methods for glycoprofiling during biologics production would fundamentally alter biopharmaceutical R&D and commercial manufacturing.
Special Lectures
Results and Discussions: The beads carrying high-density probe DNA achieved the lowest limit of detection (3.2 pM), which was 10 times lower than medium-density ones (37.1 pM). Moreover, the formerresulted in a sigmoid working curve with larger dynamic concentration range (38 pM ~ 625 nM) compared to the later (from 610 pM∼39 nM). These results showExoIIIaided recycling technique can significantly improve the sensitivity by a factor of56.8 to 68.4 compared to conventional nucleic acids bead assay (direct hybridization).3
Congress Overview
modified DNA probes were conjugated onto 15 µm beads via EDCactivated conjugation. The surface probe DNA loading after target DNA recycling were quantified by flow cytometry method (Cytek DxP6; 25 mW 639 nm lasers).
Sunday, 19 May
Results: The GlycoSenseTM method is not intended to replace full-scale characterization of biologics but makes in process glycoprotein sampling and monitoring possible. In preliminary experiments, this method was used to analyze the terminal glycosylation patterns of standard glycans and glycoproteins. The results were comparable to that of other glycan analysis techniques.
Monday, 20 May
Conclusions: The GlycoSenseTM approach is a rapid, simple glycoprofiling method that requires no specialized equipment or training. It complements the current methods of glycan characterization while addressing the unmet need for real-time glycoprofiling tools.
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Multiplexed Microsphere Protease Assays and High Throughput Flow Cytometry for Drug Discovery
Figure 1. The Schematics of Exo III technique and its aided flow cytometry DNA beads assay
Development of a Glycoprofiling Method Using Multiplex Microspheres
Speaker/Author Index
ISAC 2013 Program and Abstracts
Results: We have demonstrated multiplex microsphere based protease assays and high throughput flow cytometry based screening that discovered several interesting inhibitors to the proteases. The average Z’ value for each substrate was greater than 0.7 in our screenings and hundreds of compounds were found with nominally active (hits). We will present confirmatory assays and dose response curves for the identified compounds. We will also present the key kinetic parameters (Km, kcat and kcat/Km) of a protease as measured in microsphere-based and solution phase assays. Additionally, we have performed detailed inhibition mechanim studies of these compounds by full-length and peptide FRET assays to determine whether these compounds work with the distal sites or cleavage sites, further to determine whether they are competitive, noncompetitive and uncompetitive inhibitors.
Poster Session Abstracts
Background: Glycans (complex carbohydrates) covalently attached to the surface of a protein play a significant role in the bioactivity of molecules such as therapeutic antibodies or erythropoietin. Consequently, improper glycosylation has a direct impact on the safety and efficacy of glycoproteins. The US Food and Drug Administration (FDA) requires that the glycoprofiles of all therapeutic glycoproteins (biologics) fall within certain specifications. However, the heterogeneity of glycans and their structural isomerism make their characterization a time consuming task. Currently, glycoprofiling requires several weeks for completion by highly trained personnel using specialized instrumentation and is therefore not suitable for in process monitoring. These long
Oral Session Abstracts
Loretta Yang1, Christine Leoff2,3, Robert Woods2,3 Glycosensors and Diagnostics, San Diego, CA, United States, 2 CCRC, University of Georgia, Athens, GA, United States, 3 Glycosensors and Diagnostics, Athens, GA, United States
1
Methods: Here we have developed a multiplex substrate set containing four substrates (SNAP-25 and VAMP-2 fusion proteins, a fusion protein bearing LF cleavage site, and a negative control substrate) for Botulinum neurotoxin type A & F light chains and Bacillus anthracis lethal factor respectively. The HTS platform uses streptavidin coated fluorescent suspension microsphere arrays where each microsphere population bears a unique fluorescent protease substrate. To measure protease activity, we add three proteases simultaneously, where each cleave their substrate to cause loss of fluorescence on specific microsphere populations. The HTS has been implemented in 1536-well plates containing the >350,000 compounds of the library.
Commercial Tutorials & Exhibits
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Poster Session
References: (1) Zuo, X.; Xia, F.; Xiao, Y.; Plaxco, K. W. J Am Chem Soc2010, 132, 1816. (2) Luo, M.; Xiang, X.; Xiang, D.; Yang, S.; Ji, X.; He, Z. Chem Commun (Camb)2012, 48, 7416. (3) Spiro, A.; Lowe, M.; Brown, D. Appl Environ Microbiol2000, 66, 4258.
Background: Due to their toxicity, it is of great importance to identify potential inhibitors to Lethal Toxin of B. anthracis and the Botulinumneurotoxins. We have developed a simple and efficient protease assay that enables the use of full-length protease substrates and multiplexed assays, provides high-throughput screening (HTS) for drug discovery. HTS by flow cytometry is an efficient method to discriminate from a large number of compounds and identify the potent protease inhibitors, not only because it requires small sample volumes, it is high throughput and robust, but it is also sensitive, reproducible, and accurate.
Wednesday, 22 May
Tuesday, 21 May
Jingshu Zhu1, Larry Sklar2, Bruce Edwards2, Steven W. Graves 1 University of New Mexico, Albuquerque, NM, United States, 2 Univ of New Mexico
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Congress Overview Special Lectures Saturday, 18 May Sunday, 19 May Monday, 20 May Tuesday, 21 May Wednesday, 22 May Poster Session Commercial Tutorials & Exhibits Oral Session Abstracts Poster Session Abstracts Speaker/Author Index
Conclusions: Our multiplexed microsphere protease assay combined with high throughput flow cytometry will be applicable to a broad range of proteases due to its ability to rapidly discover inhibitors from large chemical libraries. Moreover, we have demonstrated that it can also provide critical kinetic parameters that are helpful in the study of the inhibition mechanisms. Finally, the use of full length substrate in our study is a marked improvement compared to peptide-based assays, because full-length substrates more accurately mimic the complex interactions of many proteases with their natural substrates. These interactions often include binding at distal sites and potential conformational changes of both the substrate and the protease.
Additionally, there are indications that the improved bead recovery leads to higher assay sensitivity for detections of low affinity targets, which will have enormous benefits to the field of diagnostics. The acoustic trapping protocol enables automated assay preparation, generic to anybead-based flow cytometry assays. References: [1] M.B. Meza, DrugDisc.Tod., 5 (2000) 38-41 [2] M. Evander, J. Nilsson, LabChip 12 (2012) 4667-4676 [3] B. Hammarström, M. Evander, H. Barbeau, M. Bruzelius, J. Larsson, T. Laurell, J. Nilsson, Lab Chip10 (2010) 2251-2257
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Significantly Improve Bead Recovery of Flow Cytometry Assay by Utilising a New Technology Platform for Sample Incubation Maria Tenje 1 , Neil LeBlanc 2 , Mikael Evander 1 , Björn Hammarström1, Hongyan Xia3, Axel Tojo1, Sándor Belák2,3, Thomas Laurell1,4 1 Department of Measurement Technology and Industrial Electrical Engineering, Lund University, Lund, Sweden, 2 Department of Virology, Immunology and Parasitology, National Veterinary Institute, Uppsala, Sweden, 3Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden, 4 Department of Biomedical Engineering, Dongguk University, Seoul, Korea, Republic of (South) We present a novel technology platform that increasesthe performance ofbead-based flow cytometry assays. Acoustic trapping in a microfluidicformat was used to perform the micro bead processing steps prior to Luminex(Luminex Corp.) analysis. The proposed method significantly improved bead recovery, a critical parameter for assay optimisation. The microfluidic format also significantly reduced the assay time Bead based assayshas become a standard within diagnostics [1]. However, with the automated wash station protocol for magnetic beads (Tecan Hydroflex) a significant bead loss is noted during the assay steps. This increases assay cost since increased amount ofmicro beads are requested to ensure assay reliability. We have therefore developed a microscaled non-contactacoustic trapping method where the beads can be retained in a flow and sequentially be incubated with the sample andassay reagents, resulting in a significantly reduced bead loss. Acoustic trapping is a technique to position and retain beads in a non-contact mode at distinct places in a microfluidic platform [2]. The beads are drawn into a rectangular glass capillary (crosssection: 2mmx200 µm)that is docked to an800 µm wide piezo electric transducer (PZT) (Fig. 1(a)) that extends across the capillary [3].The reaction site is specified by the acoustic pressure field gradient locally generated by the PZT.The PZT drives a l/2 acoustic standing wave with a pressure minimum in the centre of the channel above the transducerwhere the beads will be trapped, (Fig 1(b)). Sample and reagents were incubated with the beads in a slow flow past the bead cluster.For the first time, we now demonstrate how acoustic trapping can be utilised as a reaction site for beadbased flow cytometry assays. The performance of the acoustic trapping assay was compared to conventional protocols using micro titre plates and an automated wash station, runningthe 6-plex Flock Monitor assay (BioVet) on vaccinated poultry sample. The beads were analysed in a Luminex flow cytometer. Bothprotocols displayed the same assay results as compared to thecontrol assay. The bead recovery for the Tecan wash station protocol was found to be~30% and the acoustic trap showed a bead recovery of > 70%, (Fig. 2).Most importantly, the acoustic trapping platform also enabled a reduction in the assay time from 2.5 hrs to only 45 min with maintained assay sensitivity.
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Navigating the Labyrinth of Regulated Flow Cytometry in Drug Development Virginia Litwin1, Jennifer J. Stewart2, Jennifer Olsen1, Joel Puchalski1, Cherie Green3, Christopher Wiwi4 1 Covance, Inc., Chantilly, VA, United States, 2Flow Contract Site Laboratory, Kirkland, WA, United States, 3Amgen, Inc., Washington, DC, United States, 4Celgene Corp., Summit, NJ, United States As a potential new drug entity progresses from the drug discovery phase to commercial launch, supporting analytical data are generated in laboratories adhering to different regulations such as Good Laboratory Practices (GLP), Clinical Laboratory Improvement Act (CLIA) regulations, or Good Manufacturing Practices (GMP). Designing, validating, and implementing flow cytometry based assays in regulated environments presents unique challenges not encountered with other analytical technologies commonly used in drug development (JIM, 363:104-119, 2011. JIM, 363:120-134, 2011). This workshop will begin with brief presentations describing the various regulated environments encountered throughout the life cycle of a new drug entity. A presentation on GLP will be followed by a discussion of CLIA regulations. This presentation will focus on how the intended use of the data influences the validation process-exploratory biomarkers and patient stratification markers will be
ISAC 2013 Program and Abstracts
Procedures (SOPs) and 3) procedures for safe operation and validation of aerosol containment systems. The impact of this Policy has been widespread, with adoption by many laboratories and Institutions outside of the NIH. The ISAC biosafety committee is drafting a revision of the 2007 ISAC Biosafety Standard to incorporate much of the NIH Policy into the International Standard. This workshop will outline the NIH Policy and also present sample questions/scenarios of real-life situations encountered by cell sorter operators and guidelines to determine the appropriate biosafety procedures and practices.
Quantitative Cytometry - Calibration and Standardization
John Nolan1, Bartek Rajwa2, Rachel Errington3, Gyorgy Vereb4, Stephen J. Lockett5, Anil Parwani6, Gustavo K. Rohde7 1 La Jolla Bioengineering Institute, La Jolla, CA, United States, 2Purdue University, 3Cardiff University, 4University of Debrecen, 5Optical Microscopy and Analysis Laboratory, SAIC, Frederick National Laboratory, Frederick, MD, United States, 6University of Pittsburgh, Pittsburgh, PA, United States, 7 Carnegie Mellon Univ, Pittsburgh, PA, United States
Lili Wang1, Robert Hoffman2 1 NIST, Gaithersburg, MD, United States, 2BD Biosciences, San Jose, CA, United States
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Use of Kinetic Imaging Cytometry to Develop Pharmacological Approaches to Cardiac Regeneration and Preservation of Contractile Function Mark Mercola Bioengineering, Sanford-Burnham Med Res Inst. and Univ. Calif., San Diego, La jolla, CA, United States Heart failure has relatively few treatment options and remains a major cause of death in the developed world. Consequently, there has been tremendous interest in developing novel therapeutic approaches that regenerate myocardium and/or preserve function. We have developed human pluripotent stem cell-based assays for cardiomyocyte regeneration and preservation of physiological function. A new instrument, the Kinetic Imaging Cytometer (KIC) developed by Vala Sciences (San Diego), served a critical function our screening by providing high throughput, cell-by-cell analysis of calcium transients. Calcium handling is central to the contractile
Poster Session Abstracts Speaker/Author Index
ISAC 2013 Program and Abstracts
Early prediction of mitochondrial perturbation (MP) and impairment of hepatocellular bile acid transport (HBAT) by novel drug candidates is becoming a critical feature in drug development. In this workshop we will describe approaches for screening and diagnosing drug candidates for their potential to cause MP and HBAT utilizing various fluorescence based platforms. We discuss the strength and limitations of various screens and provide recommendations of where to position these assays during the drug commercialization process.
Oral Session Abstracts
Biosafety standards specific for flow cytometry and in particular, cell sorting are important for ensuring the safe operation of these instruments. Recently, the National Institutes of Health (USA) has adopted an NIH Biosafety Policy for Cell Sorters for the NIH Intramural laboratories. This Policy provides direction in the following key areas: 1) design of cell sorting laboratories. 2) the creation of laboratory or instrument specific Standard Operating
George Babcock1, Padma Narayanan2 1 University of Cincinnati, Cincinnati, OH, United States, 2 Amgen, Seattle, WA, United States
Commercial Tutorials & Exhibits
Since the first ISAC guidelines published in 1997, the ISAC has provided the research community with valuable guidance to the biosafety of cell sorting. In 2007, these guidelines were updated and upgraded to standards to best reflect the importance of these procedures and policies in laboratories around the world.
Functional Analysis of Mitochondria and Transporters
Poster Session
Kevin Holmes1, Stephen P. Perfetto2 Flow Cytometry Section, NIAID, NIH, Bethesda, MD, United States, 2Vaccine Research Center NIH, Bethesda, MD, United States
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Wednesday, 22 May
Biosafety: Biosafety Policy Meets Real Life Scenarios
Tuesday, 21 May
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Long term and cross laboratory collaborative studies require appropriate calibration and standardization protocols. This workshop will concentrate on standards and reference materials for both instrument calibration and assay calibration/standardization. The workshop will summarize current state of standards and reference materials, as well the gaps to be filled and needs remaining for cytometry standardization. The workshop will have a series of presentations followed by discussion, with the aim of defining goals for a collaborative study with which assay calibration and standardization can be demonstrated through the use of standards, reference controls, and a validated assay procedure.
Monday, 20 May
At the workshop we will present and describe the goals of the ISAC/ CYTO University program. We will specifically detail aspects for the current plans for image-based cytometry online education. Thus the workshop includes an overview of current microscopic imaging modalities and techniques, a review of current training programs in microscopy (live and online), as well as a review of other online resources, amongst other topics. The workshop will provide an opportunity for all interested parties to participate in discussions to shape the curriculum and methods for image cytometry education.
Sunday, 19 May
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The Delivery of an Image-based Cytometry Education Resource at the ISAC University
Saturday, 18 May
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ISAC’s CYTO University is an exciting new endeavor where the emphasis is to develop the GOTO online cytometry education portal, supported and contributed by experts and superb educators at the society. This workshop is open to everyone who wants to contribute to this endeavor. The aim at ISAC/CYTO U is to develop a comprehensive online education portfolio, one area of which is focused on image cytometry where the goal is to provide a learning environment to teach the fundamental concepts and tools for undertaking quantitative cell/tissue based analysis using image derived data. In short, our aim is to provide education on ‘how to convert images into parameters’ that describe the underlying biology. While there is a plethora of educational materials available regarding microscopy with linked applications in cell biology currently available online, it is clear that an integrated peer reviewed, online resource centered around image-based cytometry education is currently lacking.
Special Lectures
Attendees at this workshop will learn how flow cytometry is used in regulated environments and have the opportunity to share their experiences and challenges in this arena. In addition, participants will learn how adhering to the regulations can increase the quality of their data.
Congress Overview
used as examples. Finally, the application of GMP for cellular therapeutics will be addressed. This presentation will include the unique challenges of implementing flow cytometric methods suitable for GMP lot release and introduce the concept of the Quality by Design (QbD) approach to method characterization.
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Congress Overview Special Lectures Saturday, 18 May
performance of cardiomyocytes, and KIC was used in our screens for the automated acquisition of kinetic parameters of cardiomyocyte function. Illustrative of molecules from our studies include the first selective small molecule inhibitor of TGFbeta signaling. Mechanistic studies revealed a direct link between TGFbeta signaling and control of chromatin modifying machinery that is responsible for committing multipotent progenitors to cardiomyocyte lineage. A second example is a potential RNA therapeutic that preserves cardiac function in preclinical mouse model through targeting a microRNA that suppresses cardiomyocyte contractility.
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Non-Genetic Cell Population Heterogeneity: Implications for Cell Differentiation and Cancer Progression
Speaker/Author Index
Poster Session Abstracts
Oral Session Abstracts
Commercial Tutorials & Exhibits
Poster Session
Wednesday, 22 May
Tuesday, 21 May
Monday, 20 May
Sunday, 19 May
Sui Huang Institute for Systems Biology, Seattle, WA, United States Background: Even a clonal (isogenic) population of cells exhibits drastic cell-cell variability with respect to phenotype, including transcriptome. This non-genetic heterogeneity has a characteristic dynamics indicating that the apparently stochastic phenotype fluctuations of individual cells is not simply due to “gene expression noise” alone but instead, often involves unrecognized metastable sub-types of cells. Considering such heterogeneity changes the traditional paradigms of “molecular pathways” that is largely based on analysis of population averages, opening new vistas of cell behavior previously not appreciated. In particular, the new individual cell resolution perspective has profound implications on our understanding of cancer progression. Methods: Using a combination of transcriptomics in FACS sorted subpopulations, quantitative flow cytometry and single-cell qPCR of clonal cultures of tumor cells undergoing defined state transitions, combined with mathematical modeling, we dissect the dynamics of cell population heterogeneity and show how the “phenotypic plasticity” of cells with the same genome can affect clinically relevant processes, such as development of resistance to chemotherapy. Results: We present a series of results obtained by the integration of single-cell resolution measurements of gene expression and mathematical modeling that offer an explanation for the fractional and incomplete nature of induced state transitions (such as differentiation). We also find that the rapid development of cancer drug resistance – at least in the initial phase – is not due to selection of preexisting genetic mutants but can be induced by the drug itself, thus defying the orthodoxy of a Darwinian evolution that drives cancer progression. Conclusions: In this overview we present, using concrete examples of experimental findings, the importance of single-cell resolution measurement in the assessment of molecular changes during cell phenotype switches. The often neglected aspects of non-genetic heterogeneity coupled with high-dimensionality in cell fate changes must be considered when analyzing the response of cancer cells to therapeutics.
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Live Cell Analysis of Ncam Polysialylation in Microcommunities Using the Novel Combination of an Antibody-Mimetic EGFP-Endosialidase and the Viability Dye DRAQ7 Paul Smith1, Marie Wiltshire1, Sally Chappell1, Laurence Patterson 2, Steven Shnyder 2, Robert Falconer 2, Rachel Errington1 1 School of Medicine, Cardiff University, Cardiff, United Kingdom, 2Institute of Cancer Therapeutics, University of Bradford, Bradford, United Kingdom
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Background: Modification of neural cell adhesion molecule (NCAM) by polysialic acid (polySia) is thought to impart antiadhesive/migratory properties to cells of neuroendocrine tumors including small cell lung cancer (SCLC), offering a target to limit spread (Falconer et al., Curr Cancer Drug Targets. 2012 12(8):925). The impact of polySia-NCAM on SCLC cell proliferation is unclear given the anchorage-independent growth of cells as clusters in vitro and propensity to undergo transition to adherent forms. We hypothesized that polySia-NCAM expression enhances in vitro/in vivo proliferative capacity independent of cell adherence. To test this we developed a model SCLC system tractable for 2D/3D cell culture, functional adherence and live cell polySia-NCAM analysis. Methods: Our novel approach to tracking polySia-NCAM expression in live cells exploited a combination of EndoN-GFP (a polySia antibody-mimetic eGFP-tagged endosialidase) and the nontoxic viability dye DRAQ7. Adherent variants were successively enriched from NCI-H69 cell clusters and subjected to non-toxic removal of polySia and switching from 2D (attached) to a 3D (cluster) growth format on hydrophobic surfaces. Cells were profiled for both selective adherence to matrix components using multiple substrate arrays and polySia-NCAM heterogeneity using live and fixed cell flow cytometry and imaging. Proliferation was assessed by cell counting and Q-tracker® 705 flow cytometry. In vivo growth was determined by volume expansion of subcutaneous NCI-H69 xenografts in Balb/c immunodeficient nude mice. Results: Adherence to extracellular matrix substrates, with laminin preference, was independent of polysialylation. PolySia-negativity was not predictive for adherence. Selection for adherence resolved polySia+ and polySia- sub-populations for NCI-H69, and high polySia+ sub-populations for two other SCLC cell lines (SHP-77 & COR-L279). Highly polySia+ NCI-H69 cells showed enhanced growth rate in 2D and 3D cultures and in vivo. Growth rate of subpopulations correlated positively with polySia expression but not adherence. Surprisingly, co-culture of polySia+ and polySiacells resulted in an overall increase in the relative frequency of polySia+ cells, over several days, not attributable to cell death or differential proliferation. Conclusions: Live cell analysis of polysialylation shows modified expression profiles in NCI-H69 mixed micro-communities, revealing homeostatic influences not realised in isolated variant subpopulations. We suggest a new model of enhanced polySia expression upon SCLC cell detachment and spread with a concomitant gain of proliferative advantage. We conclude that polySia expression presents a dynamic target, independent of adherence potential, for controlling spread and tumor cell proliferation. [Acknowledgements: Grant support, EPSRC (UK) & Yorkshire Cancer Research; support studies, Dr E Furon (Cardiff) & P Cooper (Bradford); EndoN-GFP (Prof J Finne and Dr A Jokilammi, Univ of Helsinki)].
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Cytometric Assessment of Dna Damage- and mtorSignaling, the Factors Contributing to Aging and Senescence Zbigniew Darzynkiewicz, Hong Zhao, Dorota Halicka New York Med Col, Valhalla, NY, United States Background: Two conceptual models are being promoted to explain the aging phenomenon. Cumulative DNA damage caused by reactive oxygen species (ROS), by-products of oxidative phosphorylation, is one of them (ROS concept). Constitutive stimulation of the mitogen- and nutrient-sensing mTOR/S6 signaling is the second mechanism (TOR concept). Methods: The flow- and laser scanning- cytometric methods were developed to measure the level of the constitutive DNA damage/ROS- as well as of mTOR/S6- signaling in individual cells. Specifically, persistent activation of ATM and expression of γH2AX in untreated cells detected with phospho-specific Abs
ISAC 2013 Program and Abstracts
Methods: We created surrogate disease samples using an acute myeloid leukemia (AML) cell line, HL-60; labeled with CFSE. By spiking fluorescently labeled tumor cells into healthy donor blood (HD), we could work in an environment of certainty that leukemic cells were (1) present and (2) at known concentrations. Initial development experiments demonstrated limited stability (G2M were measured. A range of ±3 SD for % pHH3+ was pre-defined during development experiments.
Commercial Tutorials & Exhibits Oral Session Abstracts Poster Session Abstracts Speaker/Author Index
ISAC 2013 Program and Abstracts
Background: Aurora Kinases play an essential role in cellular division by controlling chromatid segregation. This family of proteins is often over-expressed in many human tumors and is being evaluated as anti-cancer targets with pan and selective-Aurora Kinase inhibitors.One substrate for Aurora Kinase A/B is Histone H3 at Serine10. Phosphorylation of Histone H3 (pHH3) has been associated with chromosome condensation and segregation during mitosis and meiosis, and therefore serves as a reasonable marker of the G2M position in cell cycle. To better understand the pharmacologic effects of Aurora Kinase inhibitors in vivo, we developed a sensitive flow cytometric assay to measure cell cycle and detect rarepHH3+ tumor cells in blood, using intracellular staining with anti-pHH3 antibody with DAPI (DNA content).
Poster Session
Jurkat cells were treated with 20µM caspase inhibitor(ZVAD.FMK) for 1 hour. To induce apoptosis, the cells were treated with 2µg/ ml camptothecin, 0.5µg/ml anti-CD95 antibody (anti-CD95) or 25µg/ml cycloheximide. The cells were labelled with Annexin V-Pacific Blue to detect phosphatidylserine (PS) exposure; 7-amino actinomycin D to detect the loss of plasma membrane integrity; cytochrome c-FITC to detect the release of cytochrome c from the mitochondria; tetramethylrhodamine ethyl ester (TMRE) to detect the loss of mitochondrial membrane potential; or LC3BAlexa Fluor 488 to detect autophagosome formation. Following immunostaining, single cell data acquisition was performed using the BD FACSCantoTM II.
Cherie Green1, Connie Ma2, John Ferbas2, Gloria Juan3 Clinical Immunology, Amgen, Ventura, CA, United States, 2 Clinical Immunology, Amgen, Thousand Oaks, CA, United States, 3Molecular Sciences, Oncology, Amgen, Thousand Oaks, CA, United States 1
Wednesday, 22 May
Apoptosis is a type of programmed cell death (PCD) that is crucial for all multi-cellular organisms. The cellular milieu triggers the initiation of this process, and once the cells have succumbed to the death cues they are degraded and discarded. The fact that apoptosis is a self-killing process is now well accepted. However, the question that has intrigued many researchers is how a cell commits to death and what factors instigate this decision-making process. In theory, it is believed that cells can be rescued if the apoptotic stimuli are removed and survival mechanisms are restored prior to the point-of-no-return. Empirically, however, a single event that defines the point-of-no-return in apoptosis remains to be exacted. Many would even argue that considering the complex signalling network of apoptosis, and its importance in cellular activity, it is unlikely that a single event would be the control for this process. Here, we investigate the effect of caspase inhibition on apoptosis of a leukemic T cell line.
Development and Qualification of a Whole Blood Assay to Monitor pHH3 and Cell Cyclein Patients with Acute Myeloid Leukemia (AML) Treated with Aurora Kinase Inhibitors
Tuesday, 21 May
Rubina Pal, Yaw Ohene-Abuakwa, Tim Rutherford, Frances Gibson St George's Univ of London, London, United Kingdom
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Monday, 20 May
Dissecting the Intricate Network of the Cell Death Machinery: Simultaneous Detection of Apoptosis and Autophagy Using Flow Cytometry
Sunday, 19 May
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Saturday, 18 May
Conclusions: Although the primary target of each on these agents may be different the data are consistent with the downstream mechanism in which the reduction of translation rate through mTOR/S6K signaling is coupled with a decrease in the energy production through oxidative phosphorylation and leads to a decline in the level of ROS, mitochondrial potential and oxidative DNA damage. The decreased rate of translation induced by these agents may slow down cells hypertrophy and alleviate other features of cell aging/senescence. The reduced oxidative DNA damage is expected to lower predisposition to neoplastic transformation which may result from defective DNA repair at the sites coding for oncogenes or tumor suppressor genes. The data suggest that combined assessment of constitutive γH2AX expression, mitochondrial activity (ROS, ΔΨm) and mTOR signaling provides an adequate gamut of cell responses to evaluate effectiveness of suspected gero-preventive agents.
Caspase inhibition completely attenuated anti-CD95 induced apoptosis. 4, 24 and 48 hours post-treatment with ZVAD.FMK and anti-CD95, the cells revealed no PS exposure, release of cytochrome c, or loss of mitochondrial membrane potential. However, ZVAD.FMK merely delayed the process in camptothecin or cycloheximide treated cells as the above events were detectable after 24 or 48 hours. The initial inference of this observation is that caspase activation is an upstream event in the death receptor pathway, and hence blocking caspase activation restores cell viability. However, further experimentation revealed that Jurkat cells might be protected from apoptosis by the cell survival mechanism, autophagy. Autophagy detection using LC3B-Alexa Fluor 488 revealed that the cells that remained viable, after treatment with any one of the apoptosis inducers in the presence of ZVAD. FMK, were LC3B positive. The effect of simultaneous inhibition of caspase activation and autophagy is currently under investigation. A discussion of the methods of immunostaining and these results will be the focus of this presentation.
Special Lectures
Results: The reported gero-preventive agents: rapamycin, metformin, 2-deoxyglucose, berberine, resveratrol, vitamin D3 and aspirin, all decreased the level of constitutive DNA damage signaling as seen by the reduced expression of γH2AX.They also decreased the level of intracellular ROS and mitochondrial transmembrane potential ΔΨm, the marker of mitochondrial energizing. All these agents also reduced phosphorylation level of mTOR, RP-S6 and 4EBP1 in A549, TK6, WI-38 cells and in mitogenically stimulated human lymphocytes. The most effective was rapamycin.
Congress Overview
reports constitutive DNA damage induced by endogenous ROS.The phosphorylation of Ser235/236-ribosomal protein (RP), of Ser2448mTOR and of Ser65-4EBP1 informs on constitutive signaling along the mTOR/S6 pathway.
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Congress Overview Special Lectures Saturday, 18 May Sunday, 19 May Monday, 20 May Tuesday, 21 May Wednesday, 22 May Poster Session Commercial Tutorials & Exhibits Oral Session Abstracts
Conclusion: We have developed a robust biomarker assay to monitorpHH3 and cell cycle in blood. This assay provides a valuable analytical tool to understand pharmacodynamic effects of Aurora Kinase inhibitortreatment in patients with hematologic malignancies.
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Understanding Subcellular Filament Networks from Fluorescence Microscopy Images Gustavo Rohde1, Saurav Basu2, Jieyue Li2, Kris Dahl2, Robert Murphy2 1 Carnegie Mellon Univ, Pittsburgh, PA, United States, 2Carnegie Mellon University, Pittsburgh, PA, United States Background: Network-like filament distributions play important roles in eukaryotic cells. These include cell motility, wound healing, cancer (including metastasis), intra cellular transport, and many other phenomena.Microtubules (tubulin), for example, are often the target of cancer treatment drugs (taxol) given their crucial role in cell reproduction. Detailed study of subcellular distributions has often ben hindered by the lack of appropriate imaging technologies. High-resolution alternatives (e.g. electron microscopy) are capable of extracting information pertaining to relatively small subcellular regions. However, they are poor options for high content, high throughput quantitative studies. Fluorescence microscopy (e.g. confocal) are good alternatives for high throughput studies. However, their resolution is often not high enough to obtain detailed measurements pertaining to filament distributions. Method: Here we describe a suite of techniques based on computational analysis of fluorescence microscope images that enables one to study in detail quantitative properties (e.g. number of filaments, curvature profiles, length distributions, subcellular distribution, etc.) of filament networks in 2D and 3D confocal fluorescence images of live and fixed cells. The software takes as input raw images, and based on linear filament modeling techniques, output the desired quantities. More specifically, the idea is to consider all possible line (tube)-like structures within the geometry of the image being used, and from these determine which is most likely to have been present given the measured image data available. In instances when filaments are sparsely distributed relative to the image resolution, direct estimation is possible. In instances where image resolution is too limited for direct estimation, indirect estimation of many important parameters is still possible. Results: We present results showing that parameter (number of filaments, lengths, etc.) extraction pertaining to both actin and microtubules is possible in both live and fixed cells. More specifically, we use the system to understand effects of the presence of carbon nanotubes in the actin distribution of cultured cells. We also present results demonstrating the application of our method to understanding differences in microtubule distributions of different cell types from 2D fluorescence microscopy images. Finally, given a set of many images where both vesicular proteins and microtubules are visible for the same cell, we also show that our modeling approach can also be used to study the relationship of arbitrary vesicular proteins in terms of association to filament subcellular filament networks.Amongst other applications, we utilize data from the Human Protein Atlas to show that the framework can be used to identify proteins that could be potential cancer biomarkers.
Speaker/Author Index
Poster Session Abstracts
Results: All acceptance criteria were met for this qualification. Experiments with unconjugated anti-pHH3, isotype control, and blocking peptide demonstrated specificity. Samples that were held at -70°C in 90% methanol were stable for up to 4 months.Intra- and inter-assay precision for samples with greater than 25 pHH3+ cells was less than 25% CV across triplicates and repeat draws.
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New Approaches to Study Neuronal Connectivity in A High-Content Format Natalie Prigozhina, Jordan Seldeen, Fabio Cerignoli, Ranor Basa, Jeffrey Price, Patrick McDonough Vala Sciences, Inc., San Diego, CA, United States Proper neuronal function is critical for overall physiological function, health, and cognitive abilities. Disorders involving altered neurotransmission and neuro-circuitry are debilitating to individuals, costly to society and include inherited mutations (e.g., fragile-X, Huntington’s disease, multiple sclerosis, and Parkinson’s disease), psychological disorders (e.g., autism, schizophrenia, bipolar disease) and dementia (e. g., Alzheimer’s and old age). Furthermore, damage to the brain or spinal cord due to accidents or strokes results in impaired neurotransmission and deterioration of neural networks. Additionally, a phenomenon termed “chemobrain’ reported since the late 1980s, involves a decline of learning and cognitive function, likely related to altered neurotransmission, which occurs in patients receiving anti-cancer drugs. The potential mechanisms that cause this disruption remain largely unknown, but may involve the disruption of adult neurogenesis and damage to existing normal mature neurons in the brain. Unfortunately, there are few model systems for use in developing therapeutic medications or treatment strategies for these afflictions. They primarily rely upon performance of electrophysiological measurements, done on an individual cell basis at low throughput and at high expense. The goal of our research is to develop highcontent, high-throughput methodology for testing the effects of compounds and genomic constructs on neuronal function synaptic transmission, establishment of neuronal networks, neuronal survival and the differentiation of neuronal precursor cells. Here we would like to present our in vitro assay system to quantify the effect of test compounds, both long term and acute) on neuronal transmission. We are developing an in vitro neuronal system, where the neurons spontaneously form functional synapses and exhibit spontaneous or induced synchronized activity. We then assay the activity of the neurons using fluorescent calcium and/or voltage indicators in the presence or absence of known pharmacological agents in order to identify positive and negative controls. The 10 to 20 second time-lapse movies are recorded at video rate using Kinetic Image Cytometer (KIC) and analyzed by automated image analysis software CyteSeer (both developed by Vala Sciences). We successfully cultured, imaged and analyzed primary rat hippocampal and human iPSC-derived neurons in a high throughput format (384 well). Our preliminary results demonstrate that neurons form interconnected neuronal networks and exhibit occasional spontaneous synchronized flashes meaning they form functional synapses with each other. We also showed that the activity of the neurons in our system can be modulated (inhibited or activated) by long or short term treatments with pharmacological drugs. Finally, we have correlated the calcium based activity assessment with expression and colocalization of pre- and postsynaptic markers using immunolabeling technique.
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Addressing Uncertainty in the Automated Evaluation of Stem Cell Colony Quality Michael Halter1, Ya-Shian Li-Baboud1, Adele Peskin1, Peter Bajcsy1, Daniel Hoeppner2, Anne L. Plant3 1 NIST, Gaithersburg, MD, United States, 2Lieber Institute for Brain Development, Baltimore, MD, United States, 3NIST Laboratories that culture pluripotent stem cells must make subjective decisions about which colonies to passage and which
ISAC 2013 Program and Abstracts
Sample pre-enrichment is a common process strategy in cell sorting applicationswhich is used to reduce the complexity of the sample and reduce overall sorting time.However, it can still be a timeconsuming procedureand may compromise biological function.1 Here we demonstrate an in-line sample pre-enrichment modular tool on a modified BD Influx™ cell sorter combining magnetic cell separation and acoustic cell concentration technologies2 to preprocess the sample immediately before injection into the flow cell (Figure 1).This new pre-enrichment process can speed sorts 3–4 timesoverall and specifically eliminates the time that cells sit after pre-enrichment which greatly reduces cell-cell aggregation and subsequent additional losses due to coincidence and doublets. Treg cells were directly sorted from a PBMC sample and from the same sample which had been pre-enriched for CD4 T lymphocytes using the BD IMagTM CD4 T Lymphocyte Enrichment Kit using the in-line process. Without pre-enrichment, the rateat which the sorted cells were collected was observed to be 244 cells/sec (total over-threshold event rate of 4,000 events/sec) while with preenrichment the sort rate was increased to 940 cells/sec(total overthreshold event rate of 10,000 events/sec). This higher rate allowed 1.2x106 Treg cells to be sorted in about 24 minutes or about 3.5 times faster than the un-enriched sort procedure. Waste fluid from the acoustic concentrator was subsequently analyzed and found to contain about 7% of the total cells coming from the magnet. Typical cell loss during the concentration step varies depending on sample concentration and flow rate through the concentrator and ranges typically from 1%–10%.
Wednesday, 22 May Poster Session Commercial Tutorials & Exhibits
The magnetic depletion step removes sample components prone to aggregation, such as monocytes, and improves the spatial distribution of cells immediately prior to sorting. Aggregate removal is shown in Figure 2 using a BD Influx™ Sort Analysis Tool software application which displays the position of all cells in the sample stream, calculates the proximity of the nearest neighbors, andadditionally expresses a measure of dispersion as the Entrainment Factor (EF = observed frequency / expected frequency). When EF = 1, the sample distribution can be described by normal Poisson distribution, >1 indicates aggregation and1, loss of processed events occurs due to more frequent electronic aborts and reduced sort efficiencies on the processed events due to coincidence within drop packets.
Oral Session Abstracts Poster Session Abstracts
We have demonstrated that the in-line enrichment modular tool on the BD Influx™cell sorternot onlycuts down the time to sort large
Speaker/Author Index
ISAC 2013 Program and Abstracts
Brian Warner1, Liping Yu1, Joe Trotter1, Maria Jaimes1, Marko Blom2, Wilfred Buesink2, Andreas Lenshof3, Thomas Laurell3 1 BD Biosciences, San Jose, CA, United States, 2Micronit Microfluidics, Enschede, Netherlands, 3Lund University, Lund, Sweden
Tuesday, 21 May
Determining the responses of a biological system to various perturbagens is a key paradigm for understanding the basic processes that underlie that system.Automated, high content/high throughput microscopy methods offer an opportunity to efficiently perform such experiments by surveying a large population of cells at low cost. Here we describe a non-parametric, cross-validation method to determine the pairwise similarity between populations from different conditions while simultaneously considering the relationship between experimental replicates as a baseline for evaluation. Using images of Arabidopsisprotoplasts under various perturbagen conditions, we segment regions of interestto form a distribution of responses for each experimental replicate. We next calculate features to describe the subcellular pattern in each regionand construct a confidence interval describing the ability of each feature to discriminate between the distributions of samecondition experimental replicates. Using different subsets of the replicates and iteratively discarding the features with the largest
Accelerated Cell Sorting Using In-Line Sample PreEnrichment
Monday, 20 May
Gregory Johnson1, Joshua Kangas1, Alexander Dovzhenko2, Karsten Voigt2, Santosh Bhavani1, Klaus Palme2, Robert Murphy1,2 1 Carnegie Mellon University, Pittsburgh, PA, United States, 2 Albert-Ludwigs-Universität Freiburg, Freiburg, Germany
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Sunday, 19 May
A Non-Parametric Method for the Automated Discovery of Perturbagen Responses in High-Content Analysis
Saturday, 18 May
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same-condition discriminative ability, we evaluate the error of a nearest neighbor classifier between different-condition distributions. We determine there to be a difference in control versus condition when the error between different experimental conditions is statistically greater than the error between same-condition replicates. Using the same method, we evaluate hierarchical relationships between experiments to discover phenotypes present in the data. We demonstrate the robustness of this method by using images from automated widefield microscopy images to identify an initial set of chemical compounds that affect subcellular distributions of various proteins, and confirm the results with the same method by confocal microscopy.
Special Lectures
A quantitative description of stem cell colony quality was generated by first using a reference set of colony images that were assigned a quality score by expert biologists. In this case, we found that colony scoring by different expert biologists did not always provide a consistent score. Using the inconsistent scoring data, we determined the uncertainty in the colony scoring decision. Using our set of image analysis features and we examined approaches to training a classifier using the reference set of scored colony images. Interestingly, some image analysis features were more correlated with each expert biologist, suggesting that the set of image analysis features we used capture at least some of the information used when making a manual assessment of stem cell quality. Each class definition includes an uncertainty derived from the uncertainty in the expert scoring. We compare two approaches to classification: Random Forest, which is a sequential (decision tree) method, and a linear combination approach in which a set of features is applied simultaneously. We find that the linear combination method appears to be somewhat more successful at correct classification. However, the decision tree method allows us to identify features that are less important to correct classification. This work provides a quantitative and unambiguous definition of stem cell colony quality and illustrates how uncertainty can be incorporated into this definition.
Congress Overview
to discard based on their visible microscopic characteristics. To the expert in culturing stem cells, these characteristics indicate desirable properties such as pluripotency and viability and undesirable properties such as cell death and spontaneous differentiation. Because stem cell colony quality assessment is subjective, culturing these cells can be inconsistent between laboratories and even from passage to passage within the same laboratory. If an automated approach to stem cell quality assessment could be developed, consistency and reproducibility would be improved, and automation of expansion and passaging of stem cells would be made possible. In this study, we have used descriptive explanations of visual cues from stem cell experts to provide insight into the image features that are critical in identification of desirable colonies. Using this insight, we have designed image analysis algorithms to quantify characteristics of the colonies thought to be associated with quality (i.e. pluripotency) of the stem cell colony.
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Congress Overview Special Lectures Monday, 20 May
Sunday, 19 May
Saturday, 18 May
samples, but alsoincreases detection and sorting efficiency. The mitigating effects on cell-to-cell aggregation are transitory—a preenriched sample that is left to sit for some period of time (1–2hr) begins to degrade and aggregationrecurs. This suggests that time to sort after pre-enrichment is of great importance which provides a compelling argument for the advantages of the in-line procedure.
Tuesday, 21 May Wednesday, 22 May Poster Session Commercial Tutorials & Exhibits Oral Session Abstracts Poster Session Abstracts
Methods: Microflow1’score technology is a fiber-optic flow-cell with sheathless fluidics. The fiberized design ensures fixed optimal positioning of optical components. The laser and the detection sub-systems are also fiberized. It is a simple to use, self-contained FC with: low mass (10kg), small size (34cm W x 19cm D x 20cm H), safe fluid management, low power consumption (11W) with battery operation (10 D batteries), minimal sample and waste volumes (less than 3mL) and Velcro patches to fastenFC. Prepared biological samples are delivered into the flow cytometer via a 6-chamber cartridge that locks to the FC.In preparation for the ISSdemonstration, a cartridge was loaded with stress biomarkers: a customized Th1/Th2 assay (CBA multiplexed kit by BD) to support monitoring secreted cytokines, SupT1 cell line and human blood cells prepared for CD4 and CD45 surface marker detection. Thecartridge also includes calibrationmicrofluorospheresand flushing solutionsto validate performance.Samples were also characterizedwith BD’s FACSArray, our reference method. To further reduce sample volume to 500µl, analytes canbe injected with a luer slip syringe.Two Flight Models (FM) have been tested on ground using syringes and cartridges. Results: Results indicate consistency between both syringe and cartridge injection. Multiplexed cytokines assays were within 5% of the reference method. Leukocyte sub-populations were discriminated but hadsome counting discrepancies when comparing to the reference instrument.Overall, there was acceptable correlation between the FM and EQM units.
Speaker/Author Index
which isincompatible with microgravity (2). In the past, NASA has been successfully using a modified Guava FC (3) during parabolic flights. Recently, a miniature sheathless FC, Microflow1Engineering Qualification Model (EQM), also performed well during simulated microgravity (4).Microflow1, developedby INO for the Canadian Space Agency, is scheduled for a technology demonstration onboard the ISS early in 2013.
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Microflow1 a Portable Flow Cytometer for Space Travel: In Preparation for the International Space Station Demonstration Christophe RIVIERE , Ozzy MERMUT , Genevieve DUBEAULARAMEE2, Luchino COHEN2 1 Biophotonics, INO, Quebec, QC, Canada, 2Canadian Space Agency, Saint-Hubert, QC, Canada 1
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Background: Flights in space have demonstrated that weightlessness is responsible for physiological changes includingaltered immune system (1). A flow cytometer (FC) will help to monitor physiological adaptation onboard the International Space Station (ISS).Most commercial flow cytometers depend on hydrodynamic focusing,
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Conclusions: Microflow1 EQM unitis compatible with simulated microgravity and the FM units have acceptable on-ground performance. Microflow1 or its next generationmay well be selected as apermanent installationon the ISS for online biodiagnostic/monitoring of astronauts during extended space missions. 1. Williams D, Kuipers A, Mukai C, Thirsk R. Acclimation during space flight: effects on human physiology. CMAJ 2009;180:1317-23. 2. Crucian BE, Norman J, Brentz, J, Pietrzyk R, Sams CF: Laboratory outreach: student assessment of flow cytometer fluidics in zero-gravity. Laboratory Medicine 2000, 31:569-572. 3. Sams CF, Crucian BE, Clift VL, Meinelt EM. Development of a whole blood staining device for use during space shuttle flights. Cytometry, 1999;37:74–80. 4. Christophe Rivière, Sébastien Leclair, Ozzy Mermut, Geneviève Dubeau-Laramée, Daniel Provençal, Luchino Y. Cohen, Cyto2012, Leipzig, Germany, poster #461 and manuscript in preparation.
An Extremely Parallel Flow Cytometer for Rapid Cellular Analysis Steven Graves, Pearlson P. Austin Suthanthiraraj, Menake E. Piyeasena, Andrew Shreve Center for Biomedical Engineering, University of New Mexico, Albuquerque, NM, United States Introduction: Flow cytometry is an invaluable tool for cellular analyses such as the determination of DNA content, the detection of pathogens in blood, and immunophenotyping of cells.Current flow cytometers achieve particle analysis rates as high as 70,000 events per second, while delivering sample at roughly 100 µl/ minute. To increase throughput, higher analysis rates have been achieved by running four flow cytometer analysis heads in parallel.
ISAC 2013 Program and Abstracts
Congress Overview
However, new application areas requiring detection of very rare events (e.g. circulating tumor cells, hematopoietic stem cells, fetal cellsmaternal blood) require dramatically higher throughput, both in terms of volumetric delivery and particle analysis rates.
Unbound kG-1a cells focused in the acoustic node whereaskG-1a cells focused in the pressure antinode when bound to elastomeric colloids.
Poster Session
Conclusion: We demonstrate a new form of cell separation technology which uses discriminatory acoustic forces on elastomeric colloids to isolate cells of interest as a new generation technology for ultrasensitive detections and separations. 1. Herzenberg, L, et al. Sci Am. 1976. 234(3): 108-117 2. Miltenyi, S, et al. Cytom. 1990. 11(2): 231-238 3. Laurell, T, et al. Chem Soc Rev. 2007. 36: 492-506 4. Piyansena, M, et al. Anal Chem. 2012. 84: 1831-1839
Commercial Tutorials & Exhibits Oral Session Abstracts
Introduction: Fluorescence activated cell sorting (FACS) is the gold standard for rapidly detecting and separating cells withhigh fidelity. However, conventional FACSrequires copious sheath fluid and serial (cell-by-cell) analysis. Alternatively, magnetic activated cell sorting (MACS) provides batchwise separation, yet typically with less purity and limited capabilities for multi-marker selections.1,2
Results: By modulating monomer ratios and reaction conditions, elastomeric particles exhibited uniform and tunable size, density, and compressibility, allowing for precise acoustic control. As apreliminary surrogate demonstration for AACS, elastomeric particles werebound to positive acoustic contrast polystyrene beads, where the bound complexdisplaced the pressure antinode.
Wednesday, 22 May
Charles Shields IV1,2, Leah Johnson1, Lu Gao3, Gabriel Lopez1,2 1 Biomedical Engineering, Duke University, Durham, NC, United States, 2Triangle MRSEC, Durham, NC, United States, 3 Mechanical Engineering and Materials Science, Duke University, Durham, NC, United States
Elastomeric particles were synthesized from nucleation and growth of hydrolyzed multi-functional siloxanemonomers. Particles were adsorbed with streptavidin, which bound to biotinylated anti-CD34 antibodies on kG-1a leukemia cells. Only cells expressing CD34 were labeled, thus allowing discriminatory separation.
Tuesday, 21 May
Acoustofluidic Cell Sorting via Negative Acoustic Contrast Capture Colloids
Methods: A PZT excitesthe microfluidic deviceat resonance whereupon ultrasonic standing waves form across the flow cavity. Here, channel walls correspond to pressure antinodes where particles with low density and low modulusrelative to the media (i.e., elastomeric particles)focus; the centerline corresponds to the pressurenode where particles withhigh density and high modulus relative to the media (i.e., cells) focus. When cells are labeled, the complex displaces to the antinode when the primary radiation force on the elastomeric particles is greater than that on the cell. After separation, a downstream channel trifurcation allows for isolated cell collection.
Monday, 20 May
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Sunday, 19 May
Conclusions: In this work, we will present a compact affordable parallel flow cytometer prototype that retains the optical properties of conventional flow cytometry yet has a realistic path to analysis rates >1x106 events/s and sampling rates of 50 mL/min. This system represents an important technological advance in parallel flow cytometry that will be an important for the analysis of extremely rare event applications in flow cytometry.
Saturday, 18 May
Results: We have developed parallel microfluidic flow cellsby using deep reactive ion etching to introduce many acoustic focusing channels into a single silicon wafer. We have made these flow cells with up to 100 parallel channels that each supported 3 well-defined acoustically focused streams of cells, for a total of 300 streams. Furthermore, using an EMCCD camera for image analysis, we have demonstrated well-focused streams at flow rates as high as 15 mL/ min. For coupling to our optical detection system, we will present the development of a multinode acoustic flow cell that supports 16 to 32 focused sample streams. We will also present the coupling of this flow cell to our line focused laser interrogation system and our array detection system. Finally, we will present our work towards testing the integrated flow cytometer using calibration microspheres and cell samples.
Special Lectures
Methods: In short, the laws of physics preclude simply speeding up the sample delivery rate to further increase the analysis rate of flow cytometers. Therefore, we are pursuing the development of parallel flow cells coupled with highly parallel detection approaches to create a compact affordable flow cytometer that can analyze up to 1x106cells/s and samples at 50 mL/min. We used multinode acoustic standing waves to create many parallel flow streams in a single flow channel or several flow streams in multiple microfabricated flow channels. These channels are the basis of our parallel flow cells that we have coupled to a newly developed optical detection platform. This platform uses a line-focused laser in combination with an array detector (e.g. multianode PMT or EMCCD) to detect scatter and of fluorescencefrom up to a few hundred flow streams simultaneously.
Speaker/Author Index
ISAC 2013 Program and Abstracts
Poster Session Abstracts
We propose a system as aprototype for a new generation of acoustically activated cell sorting (AACS) that isa viable, and potentially superior, alternative to FACS and MACS. Acoustophoresis provides cell separation capabilities based on acoustic properties of cells and fluid.3Using elastomeric particleswith antibody affinity labels(Fig. 1), AACS can separate rare cells (e.g., circulating tumor cells)from native fluidsby acoustic radiation forces. These forces are independent of flow, allowing continuous focusing at switchable flow rates for ultrasensitive detection when coupled with fluorescence based flow cytometry.4
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Congress Overview Special Lectures Saturday, 18 May Sunday, 19 May Monday, 20 May Tuesday, 21 May Wednesday, 22 May Poster Session Commercial Tutorials & Exhibits Oral Session Abstracts Poster Session Abstracts Speaker/Author Index
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Complex Changes in Invariant Natural Killer T (iNKT) Cells in Patients with Different Clinical Forms and Treatments of Multiple Sclerosis
Immune Monitoring of Human Kidney Allografts with Biopsy Multicolor Flow Cytometry and Cytokines
SARA DE BIASI1, Milena Nasi1, Anna Maria Simone2, Diana Ferraro2, Francesca Vitetta2, Lara Gibellini1, Marcello Pinti3, Cinzia Del Giovane4, Patrizia Sola2, Andrea Cossarizza1 1 Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia, Modena, Italy, 2MS Center, Neurology Clinic, Nuovo Ospedale Civile Sant’Agostino Estense (NOCSAE), Modena, Italy, 3Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy, 4Department of Diagnostic and Clinical Medicine and Public Health, University of Modena and Reggio Emilia, Modena, Italy Introduction: The pathogenesis of Multiple Sclerosis (MS) is still poorly understood. Associations between MS and defects in invariant natural killer T cells (iNKT) have been reported, but contrasting data exist, due to methodological limitations. iNKT cells are potent cytokine producers, have immunoregulatory potential, and can be divided into functionally distinct subsets. A more detailed characterization of iNKT cell subsets is needed to better clarify their role in MS, particularly in different forms of the disease and its treatment. Among T cells, a recently described population exists that has strong proinflammatory capacity (CD8+CD161+ T cells), and is altered in MS. Aim of our study was to evaluate the amount of these cells, along with that of iNKT, in different forms of MS. Methods: We studied 66 patients followed by the MS Center (NOCSAE, Modena): 52 with a Relapsing Remitting (RR; 46 taking therapy, 6 without treatment), 9 with a Primary Progressive (PP) and 5 with a Secondary Progressive (SP) MS. 16 age- and sexmatched subjects were healthy controls (CTR). iNKT and CD8+, CD161+ cells were analyzed on fresh PBMC. CD3+ T cells were volumetrically counted using a CyFlow Counter (Partec, Germany). Then, isolated PBMC were stained with different mAbs for the analysis on a 6-color high speed acoustic focusing flow cytometer (Attune, Life Technologies, USA), using different mAbs, i.e. antiVa24Ja18 TCR, -CD4, -CD8, -CD161, -CD3, -CD19 and -CD14. A minimum of 5 million cells were acquired, and data analyzed by FlowJo 9.6 and Stata 11.0 softwares using Ranksum and Anova test. Results: In comparison with CTR, MS patients with a RR form displayed a statistically significant lower number (but not percentage) of iNKT cells. Patients with other MS forms (whose number was lower) showed a similar trend. Among iNKT cells, the CD4+ subset did not vary in the different forms or in comparison to CTR, while the CD8+ subset was always diminished. The CD4CD8- subset was significantly lower in the RR form compared to CTR. Among iNKT cells, the population of CD8+CD161+ cells was lower in all MS patients. When compared to CTR, PP and SP patients had a lower number and percentage of CD8+ T cells; CD8+, CD161+ T cells (both dim and bright subset) were lower in all SP patients vs. CTR. Concerning the role of MS treatment, RR patients taking Fingolimod (a drug that blocks the sphingosine-1-phosphate receptor S1PR1 and traps lymphocytes in the lymphnode) had a lower number of CD3+ T cells and a lower percentage of iNKT expressing CD4. Patients taking natalizumab (blocking alpha 4-integrin), interferon or glatiramer acetate displayed no gross changes in iNKT cells in comparison with CTR. Conclusion. The changes in iNKT cells and their subpopulations we observed in RR patients, along with the trends present in the other patients, suggest a role for these cells in the pathogenesis of MS. Functional studies are ongoing to better understand the activity of such cells.
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Kimberly Muczynski, Nicolae Leca, Susan Anderson Medicine/Nephrology, Univ of Washington, Seattle, WA, United States Background: Biopsies, the standard for assessing kidney transplants, lack information about immune processes and are often insufficient for guiding therapy. Different injuries can appear histologically identical, as “nonpecific inflammation,” creating a dilemma for treatment. For example, rejection and BK nephropathy both have infiltrating leukocytes in kidney tubules but have opposite treatments. Leukocytes in fibrotic areas can represent either resolving inflammation or active injury. Also, leukocytes circulating in the local microvasculature, which may be inciting inflammation, are lost in histology processing but retained when a biopsy is processed for cytometry. Methods: To better assess allograft inflammation, I devised a collagenase digestion protocol for reducing kidney biopsies to cell suspensions for multicolor flow cytometry which preserves epitopes of interest and used a high sensitivity Luminex platform assay to measure secreted cytokines within the tissue. Allograft leukocytes, antibody-bound endothelial cells and cytokines were assessed. Results: The following observations have been made: 1) Rejection is associated with reduction in allograft granulocytes, increased CD3 cells and a predominance of CD8 over CD4 T cells. The ratio of CD8:CD4 cells identify rejection early, before it is detectable by histopathology. 2) BK nephropathy is associated with increase in CD56+ (NK) cells and a low CD8:CD4 ratio. 3) Increased antibody on endothelial cells is associated with transplant glomerulopathy and can be used to follow clinic course. 4) IL-6 and -8 are associated with renal ischemia. High levels of IL-4 predominate in rejecting renal allografts with donor specific antibodies and complement activation. Conclusion: Cytometry, to define cells and cytokine levels within kidney transplants, reliably distinguishes harmful nonspecific inflammation from immune processes that do not require adjustments of immunosuppression. It should be used as an adjunct to traditional histopathology. Results may also define targets for more specific immunosuppressants.
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Metastatic Breast Cancer Stem/Progenitor Populations Survive Consolidation Chemotherapy, Circulate and Disseminate to Bone Marrow Albert Donnenberg 1 , Vera S. Donnenberg 2 , Rodney Landreneau2, Adam Brufsky1 1 Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States, 2Cardiothoracic Surgery, Univ of Pittsburgh, Pittsburgh, PA, United States Despite the prognostic value of circulating tumor cells in late stages of breast and prostate cancers, the “stem/progenitor phenotype” and tumorigenicity of these cells have never been demonstrated. To determine immunophenotypic signatures of metastatic breast cancer cells we examined coexpression of epithelial markers and stem/progenitor markers in malignant pleural effusions from 9 breast cancer patients. Nonhematopoietic pleural effusion cells expressed cytokeratin and EpCAM, but the proportion varied widely. CD90 was the most prevalent stem/progenitor marker studied (14.5 ± 4.1%, mean ± SEM), followed by CD133 (10.7 ± 6.24%) and CD117 (3.4 ± 1.0%). These populations were largely mutually exclusive and each had significant proportions of cells with >2N DNA. Aneuploidy was limited to cells bearing epithelial markers.
ISAC 2013 Program and Abstracts
Background: Knowledge of the contextual information cells derive from their physical, chemical and cellular microenvironment within tissues is essential for the understanding and effective treatment of many diseases, especially cancer. Producing uniform, controllable in vitro 3D models of tissues and tumors is a critical requirement to advance the nascent field of tissue cytometry. Improved methods are also needed for generating 3D models containing multiple cell types, as well as for in situ assay of cells and microenvironments. Introduction: Our approach to addressing these limitations involves the generation and assay of spherical cellular aggregates (spheroids). We have engineered a novel apparatus that utilizes fluid flow and ultrasonic vibrations to produce uniform populations of calcium alginate microspheres (CAMs) containing viable cells. Proliferation of cells in suspension-cultured CAMs results in the generation of completely cellular spheroids of uniform size and cellular composition.
Monday, 20 May
Methods: Our apparatus consists of a pressure chamber producing stable flow of a sodium alginate pre-gel solution containingfluorescently labeled human tumor cells through a precisely fabricated glass micronozzle. The micronozzle is coupled to a piezoelectric transducer that produces vibrations that result in the formation of cell-containing microdroplets. The microdropletscrosslink upon interaction with calcium ions in a receiving solution, producing CAMs. Droplets are visualized through a 10x objective coupled to a camera and an LED array that strobes atthe vibration frequency. For initial interrogation of the microenvironment, the pre-gel solution is functionalized with pH-sensitive fluorescein. CAMs and the resultant spheroids are assayed both in situ by confocal microscopy and by flow cytometry following dissociation to single-cell suspensions.
Tuesday, 21 May Wednesday, 22 May
Results and Conclusions: Varying the flow rate and PZT modulation frequency controls the size of CAM populations. We can produce uniformly sized CAMs from 100-250 µm diameter at frequencies of 10-30 kHz, with initial cell concentrations up to ~25% of tissue cellularity. Assay of the spatial distribution of cellswithin CAMs during subsequent culture demonstrated proliferation and the development of cell-cell and cell-matrix interactions. CAMs and spheroids were dissociated to single cells for flow cytometry analysis of proliferation, viability and quantification of ratios of different cell types in co-culture. Functionalized, pH-sensitive CAMs containing cells will be used to monitor pH gradients created by cellular metabolism. This technology impacts the field of tissue cytometry by providing a high-throughput method for creating uniform 3D tissue models. Adding other integratedfluorescence sensors (glucose, lactate, oxygen) will create a complex but quantifiable microenvironment that will greatly improve tissue cytometry applications andour understanding of tissue and tumor biology.
Poster Session Commercial Tutorials & Exhibits Oral Session Abstracts Poster Session Abstracts
Recent studies provide strong evidence for a role for B cells in the pathology of RRMS. In order to explore the effect of IFNβ-1b treatment on B cell phenotype and function in RRMS patients, blood was drawn from 10 RRMS patients before treatment and again 2 and 6 months after injections every other day of IFNβ-1b. Cryopreserved PBMCs were thawed and stained with 10-color panels of antibodies against B cell surface antigens. At baseline, RRMS patients have increased frequencies of naïve CD19+CD20+ B cells and decreased frequencies of memory B cells when compared with age-matched healthy controls. Treatment alters the composition of circulating B cell subsets, leading to an increase after six months in circulating naïve B cells and a decrease in both memory and B1 cells, both cell types of which are potentially pathogenic in RRMS.In addition, B1 cells from RRMS patients showed a higher level of CD5 expression, which may indicate a higher level of regulatory B cells in this population. Furthermore, several phenotypic differences were seen between RRMS patients and healthy controls, including significant differences in IgM, CD95, SIGLEC-10, CD24, CD307d, PD-1 and CD305 expression. The balance of these phenotypic differences indicates a B cell population in RRMS patients that is more inflammatory and less able to be regulated. Although the number of subjects in this study was limited, these findings suggest that alterations in B cell phenotype and function may be a mechanism by which IFNβ-1b reduces disease activity.
Jacqueline De Lora, Daniel Kalb, Alice Martinic, Antoinette Trujillo, Travis Woods, Andrew Shreve, James Freyer Center for Biomedical Engineering, The University of New Mexico, Albuquerque, NM, United States
Sunday, 19 May
Daniel Mielcarz1, John DeLong1, Alan Bergeron2, Kathleen Smith1, Alexandra Heyn1, Lloyd Kasper1, Jacqueline Channon1 1 Microbiology/Immunology, Geisel School of Medicine at Dartmouth, Lebanon, NH, United States, 2Medicine, Geisel School of Medicine at Dartmouth, Lebanon, NH, United States
A High-Throughput Method for Creating Uniform 3D Tissue Models
Saturday, 18 May
Relapsing-Remitting MS Patients Have Significant Differences in Circulating B Cells Subset and Phenotype Compared with Healthy Controls, and IFNβ-1b Treatment Can Alter the Composition and Phenotype of These Subsets
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Special Lectures
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Congress Overview
We then attempted to detect cells coexpressing stem/progenitor and epithelial markers as rare therapy resistant populations in the bone marrow and blood of breast cancer patients. We made use of unique cryopreserved hematopoietic stem cell backup products (bone marrow n=3, leukapheresis n=4, 3 of which were used for in vivo tumorigenicity experiments) collected after high dose cyclophosphamide/etoposide stem cell mobilization. Flow cytometry was performed before and after immunomagnetic selection for EpCAM+ cells. An average of 1.3 x 109 cells was separated. Patient samples were compared to normal bone marrow (n=10) and normal leukapheresis products (n=4). Two of three patient bone marrow samples had detectable stem cell marker+ populations (CD90+, CD133+, CD117+) that were tumor specific because they were: 1) absent in normal samples; 2) cytokeratin+; 3) enriched by EpCAM separation. By the same criteria, 3 of 4 patient leukapheresis samples had CD133+ tumor cells. The data indicate that stem cell marker+ cytokeratin+ tumor cells survive high dose chemotherapy, circulate and disseminate. In order to determine the tumorigenicity of circulating tumor-stem/progenitor cells, 3 leukapheresis products were immunomagnetically separated for EpCAM+ and/or CD90+ cells. Recovered cells were admixed with adipose-derived stromal cells and injected into mammary fat pads on NSG mice (20 injections/specimen). Xenografts were monitored for 6 months, when animals were sacrificed and tested for tumor formation. Despite the presence of CD90+ EpCAM+ cells in all specimens tested, only 3 sites out of 60 injected generated tumors, all from the same specimen. Detailed genomic and histological analyses are in progress.
Speaker/Author Index
ISAC 2013 Program and Abstracts
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Congress Overview Special Lectures Saturday, 18 May Sunday, 19 May Monday, 20 May Tuesday, 21 May Wednesday, 22 May Poster Session Commercial Tutorials & Exhibits Oral Session Abstracts Poster Session Abstracts Speaker/Author Index
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Analysis and Sorting of Antigen-Specific Antibody Secreting Cells in Mixed Cultures Using the Affinity Matrix Technology Adriano Taddeo1, Hyun-Dong Chang1, Velia Gerl2, Bimba Hoyer2, Andreas Radbruch1, Falk Hiepe2 1 Deutsches Rheuma-Forschungszentrum (DRFZ), Berlin, Germany, 2Medizinische Klinik III m. S. Rheumatologie & klinische Immunologie, Charité - Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany One of the most time and labor intensive procedure in the production of monoclonal antibodies (mAbs) by hybridoma cell lines is the identification of antigen-specific antibody-producing clones within a heterogeneous cell population. Moreover, some hybridomas can lose the ability to produce mAbs by continuous culture and recloning is therefore recommended if hybridomas have been in culture for a long period of time and monoclonality or stability of the hybridoma needs to be confirmed. Indeed, despite their biological and practical significance, only a few methods are presently available for the analysis of secreted antibody on the single cell level by antibody secreting cells (ASCs). These methods may offer a valid alternative for selecting the antigen-specific cells allowing a faster and more accurate selection of the clones of interest. Here we describe a new concept for cytometry and sorting of live ASCs based on the specificity of the produced antibody. Basically, the secreted antibody is retained on the cell surface of the secreting cell, making it accessible to the powerful technologies for detection of surface markers allowing FACS analysis and live-cell sorting of antigen specific ASCs. ASCs were incubated with an affinity matrix composed of 1) an antibody binding specifically to ASCs coupled to 2) the antigen specific to the antibodies secreted by the ASCs. For this proof-of concept experiment an anti-CD138-OVA-construct (affinity matrix) was generated and anti-OVA secreting hybridoma clones were used as model of ASCs. Anti-OVA IgG1 secreting hybridoma cells were mixed at different ratio (from 2:1 to 1:10000) with aspecific cells (X63-Ag6.653 myeloma cells or anti-OVA IgG2a secreting cells). After a short incubation for allowing the antibody secretion, the anti-OVA antibodies secreted by ASC binds to the antigen brought to the surface of the ASC by our matrix. The OVA-specific ASCs were then stained with a fluorochrome labeled anti-IgG1 antibody and visualized by FACS. The affinity matrix technology allowed us to clearly show a specific labeling of cells producing antibodies with the desired specificity and the desired IgG subclass independently of the ratio of specific/aspecific cells. These results show that using the affinity matrix technology it is possible to select ASCs in an antigen-specific way. Therefore, this technology represents a useful tool for reducing the time and manipulations necessary for hybridoma selection and cloning and provides an innovative method for the analysis of secreted antibody on the single cell level by antibody secreting cells (i.e. hybridoma cells and plasma cells).
has proposed that the potency of cells used in therapies be characterized by relevant molecular properties. Yet, there have been no measures of potency developed for cellular transplantation. Hematopoietic reconstituting cells (HRC) are transplanted in the treatment of a variety of diseases. The number of CD34+ cells transferred is clearly associated with engraftment but it does not provide an indication of the potency at the level of the individual cell. There are 2 sources of HRC that are known to be superior to bone marrow HRC in terms of potency on a cell-by-cell basis: G-CSF-mobilized peripheral blood HRC and umbilical cord blood HRC. We have used an enzymatic amplification system for enhancing the sensitivity of flow cytometric analysis to assess HRC. In a previous study we have demonstrated that our analytical paradigm could detect transcription factor expression levels that correlated with granulocyte-macrophage colony forming units and erythrocytic burst-forming units (Cytometry Part A 83A:127-133, 2013). In the current investigation we studied HRCfrom bone marrow, G-CSF-mobilized peripheral blood, and umbilical cord blood in order to assess cellular potency. We chose 16 molecules for analysis including pathway molecules (such as phospho-Akt), transcription factors (such as HoxB4), a transcriptional repressor (Bmi-1), a translational regulator (Musashi-2), an anti-apoptotic molecule (Mcl-1), an ubiquitin-conjugating enzyme (UBC13), and an autophagy protein (Atg7). All of the 16 molecules have been previously associated with HRC function based on the results of experimental manipulations. Our results provide us with a measure of cellular potency based both on the expression levels of the 16 molecules and on the correlations of the molecules with each other. Additionally, we have uncovered a linear sequence of molecular associations that is related to the potency of the cells. By analyzing molecules known to be important in function, we have developed acogent measure of cellular potency. We speculate that this technology can be useful in developing measures that may be useful in assessing the transplantation of other cells for therapeutic purposes.
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Sorting of Specific Lymphocyte Populations from Peripheral Blood Progenitor Cell Products Using a Novel Micro-Chip Based Acoustophoretic Platform Anke Urbansky1, Andreas Lenshof2, Arshad Jamal3, Josefina Dykes3, Ingbritt Åstrand-Grundström3, Thomas Laurell4, Stefan Scheding3 1 Stem Cell Center, Lund University, Lund, Sweden, 2Lund University, 3Lund University, Lund, Sweden, 4Department of Measurement Technology and Industrial Electrical Engineering, Lund University, Lund, Sweden
David Kaplan1, Hillard M. Lazarus2, Nicholas M. Kaye1 1 Pathfinder Biotech, Cleveland, OH, United States, 2Medicine, Case Western Reserve University, Cleveland, OH, United States
Background: Processing of peripheral blood progenitor cells (PBPC) for clinical transplantation or research applications aims to effectively select or deplete specific cell populations. Usually, fluorescence- or magnetically-based sorting techniques are used for PBPC processing. Here, we investigated the performance of a novel microchip-based ´acoustophoresis´ technique, utilizing ultrasound for the separation of lymphocyte subsets from PBPC. By applying an acoustic standing wave field on to a continuously flowing cell suspension in a micro channel, cells can be separated depending on their physical properties, such as size and density.
Cellular transplantation is a relatively new therapeutic modality. Various stem cells and specific immune cells have been used successfully in treating a variety of clinical conditions. It is important to develop potency measures for the cells to be transplanted in order to ascertain that the cells will perform as expected. In fact, the Federal Drug Administration, which has jurisdiction over cell transplantation for therapeutic purposes,
Study design and Methods: PBPC samples were obtained from patients (n= 16) and healthy donors (n=6). Following density gradient centrifugation, cells were labelled with either anti-CD4 or anti-CD8 microbeads (Dynal) and sorted on an acoustophoresis-microchip. In parallel, control magnetic sorting was performed. PBPC samples, target and waste fractions were analysed for separation efficiency, recovery, T-cell function and progenitor cell content.
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Developing Measures of Cellular Potency for Therapeutic Transplantation
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ISAC 2013 Program and Abstracts
In recent years, flow cytometry has benefitted greatly from the recognized MIFlowCyt standards that cover key areas such as quality control, instrument performance, experimental set up and data reporting. With the emergence of image cytometry platforms that preserve the high event throughput of flow cytometry while delivering high content, quantitative imaging, this is an opportune time to open a cross-platform multi-discipline dialogue as to how MIFlowCyt and Imaging standards could be applied to image cytometry. While there are many aspects to this topic that deserve in-depth discussion and both the time and dedication outside the time constraints of this workshop, we will aim to initiate discussion and dialogue pertaining to a need to standardize data acquisition, analysis and reporting for IC in a manner akin to MIFlowCyt.
Poster Session Abstracts Speaker/Author Index
ISAC 2013 Program and Abstracts
Hans Minderman1, Andrew Filby2, Anne Plant3, Michael Halter3, Padma Narayanan4, Amanda White5 1 Rosewell Park Cancer Institute, Buffalo, NY, United States, 2 Cancer Research UK, London, United Kingdom, 3National Institute of Standards and Technology, Gaithersburg, MD, United States, 4Amgen, Washington, DC, United States, 5 Cincinnati Children's Hospital, Cincinnati, OH, United States
Oral Session Abstracts
Over the past decade, new imaging modalities have revolutionized the field of immunology at the level of systems, cellular, and molecular immunology. I will discuss our work using two-photon microscopy and calcium indicators imaged by TIRF microscopy to investigate T cell activation. The lymph node can be understood at the systems level as a filter that promotes cellular motility and responses to antigen. Under basal steady-state conditions, T lymphocytes home from the blood into the diffuse cortex of the lymph node and migrate rapidly in a random walk that optimizes the detection of antigen. T cells sample the surface of resident and tissue-derived dendritic cells, and if the specific peptide MHC is
Integrating Standards at the Interface of Flow and Image Cytometry
Commercial Tutorials & Exhibits
Michael Cahalan University of California, Irvine, UCI, CA, United States
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Poster Session
Single-Cell Approaches to Investigate the Immune Response
The cerebrospinal fluid (CSF) supports buoyancy of the cortex, and buffers it from injury. It is also the conduit in which lymphocytes traffic through the parenchyma of the cortex. Thus, CSF immune profiling can provide a useful tool to track inflammatory processes occurring in the central nervous system (CNS). For the last decade, our laboratory has gained expertise in characterizing lymphocyte dynamics in the CSF of patients with Multiple Sclerosis (MS), an autoimmune disease of the CNS. The goal for this presentation is to review our understanding of lymphocyte profiles in the CSF of MS patients and compare them to lymphocyte profiles in the CSF of patients at risk to develop MS and patients with other neurological diseases of the CNS. The presentation will also include a discussion on the limitations of characterizing lymphocyte dynamics in CSF, and provide examples of how lymphocyte profiling in the CSF may be used as a tool to guide novel therapeutic strategies.
Wednesday, 22 May
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Nancy Monson UT Southwestern Medical Center, Dallas, TX, United States
Tuesday, 21 May
Leukocyte adhesion under flow is mainly mediated by selectins and their ligands. To study the contact area (footprint) of rolling neutrophils at high resolution, we developed quantitative dynamic footprinting (qDF), a TIRF-based technique that provides nanometer resolution in the vertical z axis. Neutrophils roll over a selectin substrate in a microfluidic device. This method shows the z distance (height) of the plasma membrane or a cytosolic marker. The cell morphology can be represented as hills and valleys. Using this technique, we confirmed the existence of long tethers (up to 20 μm) and discovered slings, cell-autonomous adhesive structures. We adapted this method for multicolor use, thus enabling simultaneous visualization of surface topography and a molecule, visualized as a fusion protein or by fluorescently labeled antibody. Slings contain patches of PSGL-1, the main ligand for P-selectin. When slings peel from the substrate, each PSGL-1 patch provides resistance to peeling and slows the cell down. PSHL-1 patch spacing is consistent with the membrane content of microvilli, suggesting that multiple microvilli are pulled into the same sling. Slings wrap around the rolling cells and interact with the cell body by LFA-1 binding to ICAM-2.
Immune Profiles in the Central Nervous System: What We Know, What We Need To Know, and What It Means
Monday, 20 May
Klaus Ley Inflammation Biology, La Jolla Institute for Allergy & Immunology, La Jolla, CA, United States
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Image Cytometry of Cell Adhesion
Saturday, 18 May
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encountered, a proximal signaling cascade generates IP3 within the T cell, in turn depleting the endoplasmic reticulum Ca2+ store. Depletion triggers oligomerization of STIM1 proteins in the ER, which then physically translocate, while still in the ER membrane, to the plasma membrane. STIM1 forms µm-scale puncta and triggers adjacent Orai1 channels to open, enabling a local Ca2+ influx and downstream gene expression responses. Genetically encoded Ca2+ indicators fused to the cytosolic tails of Orai1 can be used to detect local Ca2+ entry. Under TIRF microscopy, the signals are sufficiently large to detect the opening of single- Orai1 channels. We are also using the dominant-negative E106A Orai1 mutant to inhibit Ca2+ influx in T cells to further evaluate functional roles in homing to lymphoid organs, chemotaxis and integrin signaling, gene expression, motility, and cell death pathways. The presentation will include highlights of these studies.
Special Lectures
Conclusion: The acoustophoresis technique can be utilized to efficiently sort bead-marked lymphocyte populations from PBPC samples with high purity and recovery without impairing lymphocyte function. Acoustophoresis is, thus, an interesting technology for PBPC processing, which has furthermore the potential to offer a single platform technique for multi-parameter cell separation.
Congress Overview
Results: The mean separation efficiency of CD4+ lymphocytes to the target fraction was 87 ± 7% with acoustophoresis compared to 95 ± 8% for control magnetic sorting (n=19). Viability of sorted cells was 95 ± 4% (acoustic) and 97 ± 3% (magnetic). Following further technical improvement of the chip, purities obtained with acoustic sorting of CD8+ lymphocytes were 96 ± 3% compared to 93 ± 4% for magnetic sorting (n=3). The median recovery of acoustically selected lymphocytes was 57%. Leukocyte subpopulation analysis performed after CD4 selection showed a relative increase of CD8, CD19, CD34, CD56, CD14 cells in the non-target fractions. These changes were due to the removal of CD4 cells and found to be within the expected range. Functional testing of sorted CD4+ cells showed unimpaired mitogen-induced proliferation capacity as well as cytokine production. Results from hematopoietic progenitor cell assays indicated a preserved colony-forming ability post-sorting of non-target cells.
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Congress Overview Special Lectures
Attila Tarnok1, Ryan Brinkman2, Vera Donnenberg3, Henning Ulrich4, Susan Vice5 1 University of Leipzig, Leipzig, Germany, 2British Columbia Cancer Agency, Vancouver, BC, Canada, 3Univ of Pittsburgh, Hillman Cancer Center, 4Instituto de Química, Univ of Sao Paulo, 5Wiley, Hoboken, NJ, United States
Monday, 20 May
Sunday, 19 May
Saturday, 18 May
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Writing, Publishing and Reviewing: Advice and Tips from Cytometry A
Scientific journals require certain quality standards from manuscripts to be acceptable for further reviewing and publication. There are some very common reasons why a paper gets reviewed and accepted or rejected. This workshop aims to highlight all major aspects of manuscript writing, submission and communication with the reviewers, points out what can (and very often does) go wrong and how to do it right in order to improve your chances to get your paper published. Special emphasis will be taken to focus on the needs for a biomedical and technical oriented journal such as Cytometry Part A including MIFlowCyt, FlowRepository and OMIP. Experts from the Cytometry Part A editorial board and from the publisher Wiley-Blackwell will contribute. Winner of the best paper award of Cytometry Part A will briefly present the winning work. Authors may bring their manuscript drafts for discussion with the editors after the workshop.
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Speaker/Author Index
Poster Session Abstracts
Oral Session Abstracts
Commercial Tutorials & Exhibits
Poster Session
Wednesday, 22 May
Tuesday, 21 May
High Throughput Flow Cytometry in Pharma Bruce Edwards1, Rob Jepras2 HSC and Cancer Center, University of New Mexico, Albuquerque, NM, United States, 2 GlaxoSmithKline, Middlesex, United Kingdom
1
Background: Flow cytometry has long been acknowledged as a technology unmatched in the ability to combine high-content measurements and analytical throughput of single cells and particles in suspension. However, only recently with the advent of high throughput sampling technology has flow cytometry’s multiexperiment throughput begun to approach the point of practicality for efficiently analyzing hundreds-of-thousands of samples. This combination of both high content and high throughput has led to the renaming of the technology as high capacity flow cytometry. Objectives: In this workshop we are soliciting input from industrial and academic laboratories that have experience with high capacity flow cytometry on topics such as: assay development and validation, techniques and tricks for efficient sample and plate preparation, perspectives on the role of high capacity flow cytometry in high content screening and systems biology, recent developments in hardware and software, favorite auxiliary equipment excluding the flow cytometer/sampling platform, and future needs and trends. Conclusions: We hope that attendees who do not use high throughput/capacity flow cytometry will leave inspired to try it and that all participants will gain new insight and ideas for both theoretical and practical approaches. Presentations will be limited to several slides to allow for extended discussion.
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Mesenchymal Stem Cell Identification and Characterization Vera Donnenberg1, Keith Wonnacott2, Albert D. Donnenberg3, Henning Ulrich4, Anne Plant5 1 Univ of Pittsburgh, Hillman Cancer Center, Pittsburgh, PA, United States, 2National Institutes of Health, Bethesda, MD, United States, 3Univ of Pittsburgh, Hillman Cancer Center, 4 Instituto de Química, Univ of Sao Paulo, 5National Institute of Standards and Technology, Gaithersburg, MD, United States
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Adult mesenchymal stromal cells also known as mesenchymal stem cells (MSC) are known for their multipotency and immunomodulatory potential. Both native and culture-expanded MSC are characterized by the expression of antigens such as CD44, CD73, CD90, CD105, and vimentin, and can be isolated from bone marrow, adipose tissue, aortic and vascular tissues. Isolated adipose derived- as well as in vitro expanded bone marrow derivedMSC are able to differentiate into a wide variety of lineages, making autologous MSC an attractive candidatefor a broad spectrum of regenerative applications. Further, their immunosuppressive potential allows the use of allogeneic as well as autologous MSC for immune-modulation. Despite the various clinical applications, we lack clear definitions and standardized procedures for the identification and quantification of MSC and evaluation of their function in order to determine the dose, purity, sterility, safety, and the potency of the implanted cells. Multiparameter flow and imaging cytometry represent methodologies uniquely suited for the detection and the monitoring of cellular products utilizing MSC. The cytometry community is poised to develop a series of validated protocols for MSC preparation, detection and monitoring using a cytometry panel design, as well as protocols for inter-laboratory comparison. Special emphasis will be taken to focus on the needs for the development and availability of validated, robust, automatable, GMP-compatible methods for the identification and isolation of human MSC. Experts from the Food and Drug Administration Cellular Therapies Branch were invited to participate, the National Institute of Standards and Technology the cytometry community and cellular products manufacturing and testing laboratories will be available for a panel discussion. The goal of this workshop is to outline a set of procedures central to assay qualification, validation and quality assessment, as well as training and qualification of personnel.
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Malaria Cytometry Howard Shapiro1, Grace Chojnowski2 1 Howard M. Shapiro, M.D., P.C., West Newton, MA, United States, 2Queensland Institute of Medical Research, Brisbane QLD, Australia Malaria kills ~ 800,000 people each year; most are children under 5 and many of the rest pregnant women. Most deaths occur in Africa and Asia and are due to Plasmodium falciparum; other parasite species (P. vivax, P. malariae, P. ovale[2 species], and P. knowlesi) are less deadly. Precise numbers of infections and deaths are uncertain because diagnoses are often made without confirming the presence of parasites in patients’ blood. Although prompt diagnosis allows cure in a few days with inexpensive drugs, emergence of resistant strains is a major concern. Insecticide spraying and distribution of bed nets help control the Anopheles mosquito vectors, and vaccines and new drugs are under development. Although high-power microscopy of well-prepared blood smears, using Giemsa’scentury-old stain, remains the most reliable method of detecting and counting malaria parasites, its success depends on well-trained observers being able to detect subtle morphological features of parasitized red blood cells, often a problem because there may be only a few such cells present on a slide, making it unlikely the microscopist will encounter even a single parasite in the time available for observation. Multiparameter flow cytometry, used for decades to detect other rare cell types in blood, e.g., stem cells and circulating tumor cells, has been viewed as too complex and costly for use in malaria diagnosis, but has recently begun to be used for research in malaria biology and pharmacology andin a few field studies of parasites at low densities.Although current flow and imaging cytometry technology promise to make apparatus substantially
ISAC 2013 Program and Abstracts
ISAC 2013 Program and Abstracts
Background: In many cancers, tumor-infiltrating lymphocytes (TILs) indicate levels of tumor immunogenicity and are a strong predictor of survival. In particular, increased levels of regulatory T cells (Tregs) are associated with poorer prognosis in some cancers. An understanding of the phenotype and spatial distribution of TILs in situ within tumor regions would be advantageous. However, visual TIL assessment cannot easily determine the type of lymphocyte in situ and multimarker quantitation is difficult with standard methods. Here we present a multi-marker, computer-aided event-counting method for determining the phenotypes of lymphocytes in follicular lymphoma sections using a multispectral imaging (MSI) and automated tissue segmentation and counting approach.
Speaker/Author Index
Introduction: Multiplexingin image cytometry remains a challenging task to implement, although the value of multiplexed biomarker analysis is increasingly recognized in tissue pathology, regenerative medicine and drug screening. Conventional image cytometers are dominantly fluorescence based systems that have limited multiplexing capability (< 4 simultaneous measurements) due to the relatively broad fluorescence emission bands. In
James Mansfield1, Chris van der Loos2, LS Nelson3, C Rose3, HE Sandison3, S Usher3, JA Radford3, KM Linton3, Richard Byers3 1 PerkinElmer, Hopkinton, MA, United States, 2Amsterdam Medical Center, Amsterdam, Netherlands, 3Pathology, University of Manchester, Manchester, United Kingdom
Poster Session Abstracts
Er Liu, Erika Duggan, John Nolan La Jolla Bioengineering Institute, San Diego, CA, United States
Phenotyping Tils in Situ: Tissue Cytometric Enumeration of Intra- and Extra-follicular Foxp3+ Regulatory T Cells in Follicular Lymphoma
Oral Session Abstracts
Multiparameter Image Cytometry Using Surface Enhanced Raman Scattering Tags and Spectral Imaging
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Poster Session
Results from studies conducted to assess the value of SCNP in probing the immune system in different clinical situations, including prediction of response to cancer immunotherapy (such as ipilimumab) in patients with metastatic melanoma will be discussed.
Conclusions: Nanoparticle SERS tags can be used for image cytometry in either an indirect or direct immunostaining format, in a manner that is compatible with concurrent fluorescence staining. We have developed a set of more than a dozen spectrally distinct SERS tags that are suitable for either spectral flow or image cytometry, allowing for highly multiparameter data to be acquired with a single excitation wavelength. The SERS tags are sufficiently bright to allow high speed data aquistion (50 msec/spectra), and imaging speed in the current system is limited by the mechanical movement of the stage.
Wednesday, 22 May
Single Cell Network Profiling (SCNP) is an approach for analyzing and interpreting post-translational protein modifications (e.g. phosphorylation, acetylation etc.) at the single cell level. This technology allows for simultaneous characterization of a range of critical cellular signaling networks in different cell subsets in complex tissues andthe effects of therapies on signaling networks. Using viable cells, measurements are made on endogenous proteins before and after exposure to extracellular modulators such as growth factors, cytokines or drugs which are chosen to evoke cellular responses that echo how the signaling system is normally, or abnormally, patterned. The proteomic readout in the presence or absence of a specific modulator is termed a “signaling node”. Signaling nodes are evaluated within cells from samples that have associated relevant clinical information, for instance regarding response to the therapy of interest. Multivariate analysis can then be performed to create predictive models that can be validated in subsequent independent studies.
Tuesday, 21 May
Alessandra Cesano Nodality, Inc., South San Francisco, CA, United States
Results: Indirect immunoSERS of cells labeled with primary antibodies against individual cell surface makers showed excellent concordance with indirect immunofluorescence (Pearson correlation coefficient 0.88). Spectral imaging (spot scanning, 50 msec/spot) of SERS tags on antibody capture beads produced spectral images that confirmed our ability to resolved five different SERS tags from a single spectral image (with p90% in the UV-C spectral region, allowing the almost interference-free irradiation of the sheath fluid as it flows through the reactor. We have installed the module in a FACS Diva cell sorter and have connected it to the sheath fluid tubing close to the nozzle. At a sheath pressure of 30 psi, we were able to reduce the number of microorganisms up to 1000-fold. The cell sorter remained free of microorganisms for at least 7 days after the sterilization with bleach was performed. Thus, the module allows for the easy and continuous maintenance of sterility in cell sorting. Due to the compactness and flexibility of the UV-C LED module, it can easily be customized and implemented to other existing and newly developed cell sorters.
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Hollow Core Photonic Crystal Fiber (HC-PCF) Laser Sources: Closing in on True Tunable Laser Sources for Flow Cytometry William Telford1, Veena Kapoor1, Nga Hawk1, Yingying Wang2, Frederic Gerome2, Fetah Benabid2 1 National Cancer Institute, Bethesda, MD, United States, 2XLIM Research Institute, Limoges, France Modern high-end flow cytometers can be equipped with many lasers of varying wavelengths (nine or more in some cases), allowing excitation of a wide variety of fluorescent probes. However, integrating many individual lasers into a single cytometer is a brute-force approach to the problem of exciting many fluorochromes, being both inefficient and costly. The “ideal” laser for flow cytometry would produce multiple, discrete tunable lines from a single laser source, allowing any wavelength and combination of wavelength desired. Supercontinuum white light lasers emit over most of the visible spectrum and have been previously used in flow cytometry as tunable sources. However, their continuous emission and relatively low power per nanometer require optical filters with wide bandwidths to isolate the wavelengths of interest with sufficient power to provide good excitation. True tunable fiber lasers with discrete wavelength emission (~1 nm) have also been demonstrated for cytometry but only cover narrow regions of the visible spectrum. Hollow core photonic crystal fibers (HC-PCFs) are specialized fiber optics with a hollow structured core. These fibers can be sealed and pressurized with various Raman gas mixtures at high internal pressures. When optically pumped with a powerful visible laser source, HC-PCFs emit a series of Raman laser lines with Stokes shifts both higher and lower than the pump laser wavelength. For example, a 532 nm pump laser coupled to a HC-PCF pressurized with hydrogen gas produce discrete laser lines at approximately 487, 502, 515, 550, 563, 587, 610 and 622 nm. This technology is related to the supercontinuum phenomenon; unlike supercontinuum sources, however, HC-PCF bands are discrete with bandwidths comparable to traditional single wavelength lasers (~ 1 nm), and are powerful enough for use in flow cytometry. The laser line arrays produced are quite relevant to the excitation spectra of many traditional fluorochromes. Changing pump laser wavelength and the Raman gas in the fiber produces other arrays with varying wavelengths. For example, a 514 nm pump laser exciting a hydrogen-pressurized fiber produces discrete laser lines at 530, 546 and 564 nm. In this study, several HC-PCF
ISAC 2013 Program and Abstracts
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Determining Intracellular Protein Localization with Fluorescence Lifetime-Based Flow Cytometry 2
2
2
Methods: We used the scanning flow cytometry, which is based on the measurement of angle-resolved light-scattering patterns (LSPs) of individual cells and on the solution of the inverse lightscattering (ILS) problem. We measured LSPs with the Scanning Flow Cytometer fabricated by CytoNova Ltd. (Novosibirsk, Russia, http:// cyto.kinetics.nsc.ru/).The solution ofILS problem is based on fitting an experimental LSP with theoretical ones, modelingplatelets as oblate spheroids.Thus volume and shape of individual plateletsin a sample are determined.
Poster Session Commercial Tutorials & Exhibits
In addition to measuring platelet LSPs, we also performed antibody (anti-P-selectin) labeling to correlate shape and surface antigen expression of platelets. We used several agonists (ADP, collagen, and thrombin) to induce platelet activation. Flow-cytometric measurements were accompanied by microscopic observations and Coulter analysesas independent controls.
Oral Session Abstracts
Results: The study of platelets with the scanning flow cytometry wasperformed for several donors. The platelets volume and shape determined by the solution of the ILS problem were in an agreement with the corresponding data from microscopic observation and Coulter analyses. The determined platelet shape showed a correlation with the P-selectin expression after stimulation by several agonists.
Poster Session Abstracts
Conclusions: The shape-based approach to detect activated platelets is presented. It does not require labeling step, which facilitates rapid tests in clinical setting. High precision in measurement of platelet axis ratio and volume opens a way to study the kinetic details of platelet activation. The results of the comparison with existing methods are presented.
Speaker/Author Index
ISAC 2013 Program and Abstracts
Wednesday, 22 May
Results: The fluorescence lifetime value of EGFP was changed when localized into different subcellular regions of the MCF-7 cells. The fluorescence lifetimes changed to a larger extentthan the average fluorescence intensity because the average concentration of the EGFP remained the same despite its location within the cell.When the EGFP-LC3 protein localizedinto autophagosomesand formed punctate regions during amino-acid starvation, the average fluorescence lifetime became shorter compared to the protein in a diffuse
Background: Blood platelets are involved in many diseases, including myocardial infarction, stroke, peripheral vascular disease, cancer, and many infections. Their hyperfunction, especially inappropriate platelet activation, plays a prime role in the increasing heart-disease burden of society. It is, therefore, important to assess platelet activation. There are several approaches to detect activated platelets measuring the expression of surface receptors with the flow cytometry. We propose an alternative approach based on the measurement of platelet shape, also being a marker of platelet activation. This measurement is possible with the scanning flow cytometry and does not require labeling. Hence, it is promising for clinical analyses due to performing the test instantly after the venipuncture.
Tuesday, 21 May
Methods: Enhanced green fluorescent protein (EGFP) was chosen for all protein localization studies. EGFP was expressed as a tagged partner to different proteins using a breast cancer cell line(MCF-7). The tagged constructs includedEGFP-LC3, EGFP-mLC3 (G120A, a mutant protein that is unable to localize at autophagosome) and EGFP-p27.To induce subcellular localization of the LC3 and p27 proteins, amino-acid and serum deprivation was applied. Also, nucleo-cytoplasmic, or otherdistribution of the LC3 and p27 proteins was confirmed through confocal microscopy and cellular fractionation. The average fluorescence intensity and fluorescence lifetime were measured with an Accuri flow cytometer as well as a home-built fluorescence-lifetime based flow cytometer, respectively. Finally, protein levels were measured by western blotting.
Alexander Moskalensky 1,2, Maxim Yurkin 1,2, Anastasiya Konokhova1,2, Dmitry Strokotov1,2, Vyacheslav Nekrasov1,2, Andrey Chernyshev1,2, Valeri Maltsev1,2 1 Laboratory of Cytometry and Biokinetics, Institute of Chemical Kinetics and Combustion SB RAS, Novosibirsk, Russia, 2Department of Physics, Novosibirsk State University, Novosibirsk, Russia
Monday, 20 May
Background: An important role in the normal biological activity of proteins is theirlocalizationto different regions inside the cell. Protein localization can be readily measured with microscopy using standard fluorescence or immunofluorescence-based techniques. Flow cytometry can also detect protein presence, howeversubcellular changes in protein location are difficult to measurewithoutimage-based approaches. In this contribution, we introduce fluorescence lifetime-based cytometry as a way of indicating protein movement inside the cell at a high-throughput level. That is, changes in the fluorescence lifetime of fluorescent proteins are measured when bound to other proteins of interest, whose subcellular locationsare altered. By detecting the fluorescence lifetime changes, we are able to indirectly measure two key protein localization and mislocalization events: (1) the nuclear to cytosoliclocalization of the p27 cell cycle regulator, an event which is correlated with cell cycle progression and also, carcinogenesis; and (2) the localization of the LC3 protein to particular regions of the cytosol to form autophagosomes, a subcellular characteristic of autophagy.
A Label-Free Shape-Based Detection of Activated Platelets with Scanning Flow Cytometry
Sunday, 19 May
Ali Vaziri Gohar , Ruofan Cao , Wenyan Li , Patrick Jenkins , Jessica P. Houston3, Kevin D. Houston4 1 Molecular Biology Program, New Mexico State University, Las Cruces, NM, United States, 2Department of Chemical Engineering, New Mexico State University, Las Cruces, NM, United States, 3Molecular Biology Program and Department of Chemical Engineering, New Mexico State University, Las Cruces, NM, United States, 4Molecular Biology Program and Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, NM, United States 1
Conclusion: The fluorescence lifetime is a quite powerful photophysical trait that indicates differen tchemical and biochemical environmentsof fluorophores. In the context of subcellular protein localization, we envision a powerful highthroughput technique for drug and target screening. Future work will be to evaluate the lifetime sensitivity and resolution and introduce other protein localization events. This study was supported by a New Mexico State University Interdisciplinary Research Grant by the Office of the Vice President for Research.
Saturday, 18 May
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cytoplasmic state. We observed many fluorescence lifetime changes (3-ns to 10-ns) for several different controls ranging from mutant LC3, non-starved cells, cells with only EGFP, cells with no EGFP, etc. Additionally, when the p27 protein localization was studied a range of 3 to 4 ns fluorescence lifetime changes were observed.
Special Lectures
While still an early technology, HC-PCFs have the potential to produce many useful, discrete laser lines from a single laser source, making them another step in the “any excitation wavelength, any emission wavelength, any probe” paradigm of flow cytometer design.
Congress Overview
pump laser and gas configurations were assembled as excitation sources on a BD LSR II and modified FACSort at both the NIH in the USA and the XLIM Centre in France, and used to excite a variety of fluorescent probes, including PE and APC on MESF microspheres, and cell lines expression several red fluorescent proteins including DsRed and tdTomato. In all cases, the laser lines produced from HC-PCF sources gave equivalent excitation to conventional single wavelength lasers.
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Congress Overview Special Lectures Saturday, 18 May Sunday, 19 May Monday, 20 May Tuesday, 21 May Wednesday, 22 May Poster Session Commercial Tutorials & Exhibits Oral Session Abstracts
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Kinetics of Annexin A5 Interaction With Model Membranes, Determined by Flow Cytometry
Novel Flow Cytometry Assay for Real-Time Detection of Molecular Extension of Lymphocyte FunctionAssociated Antigen-1
Nicolas Arraud, Céline Gounou, Alain Brisson CBMN, University of Bordeaux, Bordeaux, France Annexin A5 (AnxA5) is a soluble protein that binds calcium and negatively charged lipids, principally phosphatidylserine (PS) with a sub-nanomolar dissociation constant (Kd)(Tait, Gibson, and Fujikawa 1989). AnxA5 is a popular marker of apoptosis and platelet activation, processes characterized by the exposure of PS lipids on the outer leaflet of the plasma membrane. Despite extensive studies, important questions concerning the elementary interaction between AnxA5, calcium and PS are still unresolved, principally the kinetic parameters of the interaction or the very nature of the AnxA5 binding site.These questions deserve to be answered in the context of the biotechnological or biomedical applications of AnxA5. As evidenced by the pioneer work of Sklar and Nolan, flow cytometry is a unique method for investigating the thermodynamic and the kinetics of molecular interactions, provided one of the partner is fluorescently labeled and one of the partner is large enough, or fluorescent enough, to be detected.We applied to AnxA5 an approach previously developed by Gilbert and coworkers for studying the binding of coagulation factors to model membranes deposited on silica microspheres, called lipospheres (Gilbert et al. JBC, 267, 15861-8, 1992). Factor VIII functions in an enzyme complex upon the activated platelet membrane where phosphatidylserine exposure correlates with expression of receptors for factor VIII. To evaluate the specificity of phosphatidylserinecontaining membrane binding sites for factor VIII, we have developed a novel membrane model in which phospholipid bilayers are supported by glass microspheres (lipospheres. We produced a mutant of AnxA5 that contains a single cystein located oppositely to the membrane binding face, allowing the straightforward synthesis of AnxA5 labeled with a single fluorophore, using maleimide chemistry. The interaction between fluorescently labeled AnxA5 and 20% PS-containing lipospheres (1.5 µm diameter) was performed by flow cytometry using a conventional Cytomics FC 500 (Beckman Coulter). Equilibrium experiments performed at various lipospheres and AnxA5 concentrations show the very high sensitivity of this method, as binding was detected down to tens of femtomolars of protein, with a calculated Kd in the picomolar range. At low protein concentration, binding took enough time to reach equilibrium so thatbinding kinetics could be recorded, over time periods ranging from few minutes to one hour. Kinetics data were extracted using CXP software Automator. The initial binding rate was linearly dependent on both AnxA5 and liposphere concentration, indicating a bi-molecular reaction model. The dissociation process was extremely slow, almost non detectable at constant calcium concentration. The binding kinetic constants were determined. Full kineticdata were fitted to calculate kinetic constants by an independent method. This is to our knowledge the first analysis to the kinetics of AnxA5 interaction with supported lipid bilayers using flow cytometry. The information provided by this study allows to describe extensively and quantitatively AnxA5 binding to model membrane lipospheres. It is thus possible to use these parameters to assay picomolar concentrations of AnxA5 within minutes. We are currently developing applications based on this fast and sensitive AnxA5 assay, and we are extending our work for providing a comprehensive model of AnxA5, calcium and lipid interactions.
Alexandre Chigaev1, Yelena Smagley2, Shilei Zhang3, Mark Haynes2, Wei Wang3, Larry Sklar2 1 Pathology, Cancer Center, University of New Mexico, Albuquerque, NM, United States, 2Pathology, Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, United States, 3Chemistry and Chemical Biology, University of New Mexico, Albuquerque, NM, United States Background: Integrins are a large family of cell adhesion receptors widely expressed on mammalian cells. Integrin-dependent cell adhesion can be rapidly and reversibly modulated in response to cell signaling without a change in receptor expression.In the case of integrins cell adhesion is modulated through a series of conformational changes within the molecule that include changes in the affinity of the ligand binding pocket, molecular extension, and clustering, as well as byother mechanisms.The ability to detect these changes in real-time is critical for the understanding of integrin physiology. Until recently, using flow cytometry for real-time detection of integrin conformational and affinity changes was limited to the alpha4beta1-integrin (VLA-4), the adhesion molecule that is expressed on leukocytes, hematopoietic stem cells, dendritic cells, and other cell types. This was achieved using a unique set of assays, where a ligand-mimicking fluorescent probe served as a donor in a fluorescence resonance energy transfer (FRET)-based extension assay, and the fluorescent probe binding kinetics was used to determine ligand binding affinity. Here we present a set of data, where we used two novel probes specific for another leukocyte integrin, namely Lymphocyte FunctionAssociated Antigen-1 (alphaLbeta2-integrin, LFA-1), to detect real-time conformational extension on live cells at natural receptor abundance. Methods: One of the distinctive features of LFA-1 is the inability to bind LFA-1 specific ligand in the absence of “inside-out” activation. Therefore, it was impossible to use a ligand-mimicking probe asa FRET donor as was previously done for VLA-4 integrin. We took advantage of the two published structures of LFA-1-specific allosteric antagonists that can bind to LFA-1 without cellular activation (BIRT0377 and XVA-143). Fluorescent derivatives of these molecules served as FRET donors and the red fluorescent lipid-like molecule (PKH 26) served as the FRET acceptor. Results: Because the distance between the two ligand-binding sites for the two probes iscomparable to the Forster distance for the two fluorophores, we detected a significant difference between the quenching kinetics for BIRT0377- and XVA-143- based probes. Very rapid signal quenching detected for the XVA-based probe suggests thatat rest theXVA-143-ligand binding site is located in close proximity to the membrane. Triggering the signaling pathway frequently used for the T-cellactivation induced rapid unquenching of the FRET signal consistent with previously reported rapid extension of the LFA-1 molecule. Surprisingly, the change in the signal was significantly larger than previously reported for VLA-4 integrin. Conclusions: This novel assay for detecting of LFA-1 extension can be used for real-time studies of the integrin physiology, since molecular extension is believed to be a part of the normal mechanism of integrin activation, which regulates cell recruitment and mobilization.
Speaker/Author Index
Poster Session Abstracts
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ISAC 2013 Program and Abstracts
Discovery of Regulators of Receptor Internalization by High Throughput Flow Cytometry
Results: SERS-stained capture beads were measured using either 635 nm excitation in a conventional flow cytometer or 660 nm excitation in a custom spectral flow cytometer. All SERS tags showed similar binding behavior to the calibrated bead set: At low capture densities, SERS intensity increased linearly, giving a measure of the brightness per tag. At higher capture densities, SERS intensity plateaued as steric hindrance prevented the binding of additional tags. Using the per tag brightness estimates obtained from low density beads, the total brightness at saturation, and the surface area of the capture beads, we calculated the effective binding foot print of the different SERS tags. We found that SERS
Poster Session Abstracts Speaker/Author Index
ISAC 2013 Program and Abstracts
Methods: We prepared beads (3 um diameter) with different amounts of capture molecules (neutravidin or anti-IgG) and used fluorescent ligands (biotin-PE or IgG-PE) and calibrated intensity microspheres (QuantaBrite PE) to determine their binding capacity. We prepared several different formulations of SERS tags from nanorods or nanoshells of Au and/or Ag, all with a red resonance (excitation maximum), and functionalized them with biotin or IgG. These SERS tags were characterized by TEM and nanoparticle tracking analysis (NTA), as well as Raman and UV/vis spectroscopy. We than labeled the calibrated capture beads with the functionalized SERS tags, and measured their SERS intensities using conventional and spectral flow cytometry.
Oral Session Abstracts
Interaction between antigen-specific T cells and antigen presenting cells (APC) cognate ligand involve reorganization of the cytoskeleton and recruitment of adhesive and signaling molecules to the site of intercellular contact. Sustained adhesion of T cells to APCs and formation of the immunological synapse after T cell receptor stimulation are required for the antigen-specific response.
Introduction: Nanoparticle tags exhibiting surface enhanced Raman scattering (SERS) offer an important complement to fluorescence labels for analytical and cytometry applications. SERS tags can be tuned to excite at specific wavelengths, encoded with distinctive spectral signatures, and functionalized with recognition molecules. Moreover, SERS tag photostability make them useful for imaging, and their tunability in the near-IR make them attractive for in vivo use. As for fluorophores, brighter SERS tags are nearly always better, and designing and producing SERS tags with increased brightness is an area of active research. A limitation in the field is a lack of standardized and transportable methods for characterizing the performance of SERS tags. Adapting an approach from fluorescence flow cytometry, we prepared capture beads with known binding capacity and used these to estimate the brightness and binding footprint (size) of several different SERS tag varieties.
Commercial Tutorials & Exhibits
Haley Pugsley, Sherree Friend, Raymond Kong, Brian Hall, Shobana Vaidyanathan, David Basiji Amnis part of EMD Millipore, Seattle, WA, United States
John Nolan1, Erika Duggan2 La Jolla Bioengineering Institute, La Jolla, CA, United States
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Poster Session
Studies of Immunological Synapse Formation and Downstream Signaling Events Using the FlowSight and ImageStream Imaging Flow Cytometers
Development of Brighter Surface Enhanced Raman Scattering Tags for Multiplexed Cytometry
Wednesday, 22 May
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Tuesday, 21 May
Conclusions: These findings suggest that the platform is suitable to search for receptor ligands with diverse and previously unknown efficacies. This approach has also been advanced to simultaneously monitor the trafficking of two receptors in a single cell, which provides a unique toolset to study the behavior of receptor/coreceptor pair due to drug synergy effects.
Monday, 20 May
Results: Primary screening yielded several new compounds as hits in addition to all known drugs for the β2AR in the library.A series of biochemical and biological assays including the FAP based approach was carried out to discriminate canonical and non-canonical ligands and to investigate the signaling pathways associated with these new hit compounds. Along with several weak orthosteric agonists, a compound that regulated surface β2AR expression via an unknown mechanism was identified. These results are reported in a recent publication (Wu et al, 2012. Mol Pharm, 82, 645-657).
Sunday, 19 May
Methods: We present a high-throughput flow cytometry compatibleapproach to discover new ligands that regulate receptor trafficking, and to identify their specific signalling pathways. The β2adrenergic receptor (β2AR) was chosen to be our model system.The human β2AR with a fluorogen activating protein (FAP) tag at the extracellular N-terminus was stably expressed in human monocyte U937 cells that are accessible to the fluorogen Thiazole Organge (TO1-2p). A high-throughput flow cytometry screen against the Prestwick Chemical Library (PCL) containing ~ 1200 off patent drugs was conducted
Saturday, 18 May
Background: Cell surface receptors such as G-protein coupled receptors (GPCRs), receptors for tyrosine kinases (RTKs), and ion channels are major drug targets because they play an important role in both intercellular target activation and intracellular communication. Ligand induced trafficking of these receptors can serve as a useful therapeutic model. However, direct measurement of plasma membrane protein trafficking by flow cytometry has been challenging.
One way to measure an immunological synapse is by fluorescently labeling the molecules that have been recruited to the synapse and imaging by fluorescence microscopy. However, immunological synapses are rare and therefore difficult to analyze objectively and statistically by traditional microscopy methods. To overcome these problems, we employed the Amnis imaging flow cytometers to objectively collect imagery of large numbers of cells. We report the percentage of T cells involved in an organized immunological synapse, the recruitment of adhesion molecule LFA-1 and signaling molecule Lck to the synaptic complex and subsequent translocation of NFkB from the cytoplasm to the nucleus in the T cell. In this study, Raji B cells loaded with Staphylococcal enterotoxin B (SEB) were incubated with human T cells to create T cell-APC conjugates. Cells were stained in various combinations for CD3, CD19, Actin, LFA-1, Lck and NFkB. Results from the FlowSight and the ImageStream imaging flow cytometers are compared. Using the FlowSight imaging flow cytometer we demonstrate image-based parameters that were used to assess the frequency of conjugates with an organized immunological synapse in an objective and statistically significant manner. Employing the ImageStream imaging flow cytometer we further evaluate the specific location of the adhesion and signaling molecules LFA-1 and Lck within the immunological synapse complex in T cells and measure the nuclear localization of NFkB in the T cell.
Special Lectures
Yang Wu1,2, Phillip Tapia3, Gregory Fisher4, Alan Waggoner5, Jonathan Jarvik5, Larry Sklar1,6 1 Pathology, University of New Mexico, Albuquerque, NM, United States, 2Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, United States, 3UNM Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, United States, 4Technology Center of Netwarks and Pathways, Carnegie Mellon University, Pittsburgh, PA, United States, 5Department of Biological Science and Technology Center of Netwarks and Pathways, Carnegie Mellon University, Pittsburgh, PA, United States, 6 Center for Molecular Discovery, University of New Mexico, Albuqerque, NM, United States
Congress Overview
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Congress Overview Special Lectures Saturday, 18 May Sunday, 19 May
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