AttuneTM Acoustic Focusing Cytometer Training Manik Punj Attune Training

Attune Training Agenda Section 1

An Introduction to Flow Cytometry

Section 2

An Introduction to Acoustic Focusing Hydrodynamic Focusing vs. Acoustic Focusing

Section 3

Instrument Systems Optics, Fluidics, Electronics

Break Section 4

Performance Tracking

Section 5

Software Overview Ribbons and Menus

2 | Life Technologies Proprietary & Confidential | 11/7/2011

What is Flow Cytometry? CYTOMETRY is the measurement of physical or chemical characteristics of cells or particles FLOW CYTOMETRY measurements are made as cells or particles in suspension pass individually through a flow cytometer instrument •

Performed on single cell suspensions

•

Provides discrete measurements from each cell in the sample

•

Provides a distribution of the measured characteristics in the sample

3 | Life Technologies Proprietary & Confidential | 11/7/2011

Flow Cytometry: What Can I do? • Flow cytometric measurement records data from single cells • Rapid statistical analysis on large numbers of cells are obtained • Identifying subpopulations within a heterogeneous population • Ability to identify cell populations on multiple characteristics • Ideally suited for blood and other cells in suspension • Ability to archive data • Data format allows post acquisition analysis

4 | Life Technologies Proprietary & Confidential | 11/7/2011

Flow Cytometry Basics 1. Cells in a single profile pass through the flow cell 2. Laser hits individual cell Passing through the narrow tube called flow cell. 3. Deflected light hits a series of detectors (PMTs) 4. The signal from detectors are interpreted by a computer

Flow cell figure taken from http://www.med.umich.edu/flowcytometry/training/lessons/lesson1/index.htm 5 | Life Technologies Proprietary & Confidential | 11/7/2011

Particle Delivery: Hydrodynamic Focusing Hydrodynamic core

Laser Cross Sectional Area

particle focus = narrow distribution

−sheath

−sheath

−Count

−narrow

6 | Life Technologies Proprietary & Confidential | 11/7/2011

−Intensity

Particle Delivery: Hydrodynamic Focusing Low Flow Rate

High Flow Rate

7 | Life Technologies Proprietary & Confidential | 11/7/2011

−sheath

−sheath

−sheath

−sheath

Laser Cross Sectional Area

Particle Delivery: Hydrodynamic Focusing broad particle focus = broad distribution −Count

Laser Cross Sectional Area

Increase sample input volume = increase flow rate = decrease in pressure difference = increase core diameter

•

Particle distributions broadened

•

Instrument resolution decreased

Volumetric sample rates = 25 ul/min – 150 ul/min 8 | Life Technologies Proprietary & Confidential | 11/7/2011 •

−sheath

•

−sheath

−Intensity

Acoustic Focusing: How does Attune™ Cytometer Differ from Conventional Flow Cytometers?

9 | Life Technologies Proprietary & Confidential | 11/7/2011

What is Acoustic Focusing? A century old phenomenon

No acoustic force

10 | Life Technologies Proprietary & Confidential | 11/7/2011

With acoustic force

Acoustic Focusing Capillary ~20cm

Piezo-electric device Capillary

acoustic waves – similar to ultrasound used to visualize a fetus in utero.

Focused Particles/cells

~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Flow

•Sheath is not required to focus cells •Flow rate can also be increased while maintaining resolution •Flow rate past laser can be precisely controlled to very slow rates, allowing more fluorescence and scatter signal per particle 11 | Life Technologies Proprietary & Confidential | 11/7/2011

Laser (~10µm high)

Acoustic Focusing Low Flow Rate

High Flow Rate

Laser Cross Section

Acoustically focused

12 | Life Technologies Proprietary & Confidential | 11/7/2011

−sheath

−sheath

−sheath

−sheath

Piezoelectric Transducer

Acoustic Focusing Cytometry: Practical Considerations Capillary

Cells

Piezoelectric ultrasonic device

Laser (~10µm high)

Detector

13 | Life Technologies Proprietary & Confidential | 11/7/2011

•

Acoustic forces cause cells to line up in the center of the capillary. No sheath flow is necessary for particle alignment.

•

Flow rate past laser can be precisely controlled to very slow rates, allowing more fluorescence and scatter signal per particle (better sensitivity)

•

Faster sample flow rates and speedy analysis of dilute samples facilitates rare event analysis

Acoustic Focusing

14 | Life Technologies Proprietary & Confidential | 11/7/2011

Acoustically Focused Sample

Laser Cross Section

particle focus = narrow distribution −Count

−narrow

Acoustic focusing

15 | Life Technologies Proprietary & Confidential | 11/7/2011

−sheath

−sheath

−Intensity

Focus Cells Long Before They Get There

Variable Laser Interrogation Times

Laser Interrogation Point

Laser Interrogation Point

Variable flow speeds

Sensitivity HighHigh sensitivity mode/High sample rate Transit Time = 40 usec Total Volume = 600ul/min Velocity = ~0.5m/s 16 | Life Technologies Proprietary & Confidential | 11/7/2011

LowStandard sensitivitySensitivity mode/High sample rate mode Standard

Transit Time = 10 usec Total Volume = 2400 ul/min Velocity =~2m/s

Controlling Cell Speed Without Hydrodynamic Focus High sensitivity 100 ul/min 0.5 meters/sec

Standard sensitivity 100 ul/min 2 meters/sec

Detector Laser

Focusing Solution Manifold Piezoelectric ultrasonic device

Cells

17 | Life Technologies Proprietary & Confidential | 11/7/2011

Transit Modes and Times • Standard (2400 ul/min total volume) Pre-set Sample Input Rate (in ul/min)*

Focusing Fluid Input Rate (in ul/min)

Focusing Fluid to Sample Ratio

25

2375

95:1

100

2300

23:1

200

2200

11:1

500

1900

3.8:1

1000

1400

1.4:1

*note that the particle velocity and interrogation time remains constant regardless of the sample input rate since the total volume (sample and focusing fluid) is constant at this transit mode

• High Sensitivity (600 ul/min total volume) Pre-set Sample Input Rate (in ul/min)

Focusing Fluid Input Rate (in ul/min)

Focusing Fluid to Sample Ratio

25

575

23:1

100

500

5:1

18 | Life Technologies Proprietary & Confidential | 11/7/2011

Questions ?

19 | Life Technologies Proprietary & Confidential | 11/7/2011

Section 2 - Instrument Systems • Flow Cytometer is comprised of 3 subsystems: • Fluidics - To introduce and focus the cells for interrogation • Optics - To generate and collect the light signals • Electronics - To convert the optical signals to proportional electronic signals for computer analysis

20 | Life Technologies Proprietary & Confidential | 11/7/2011

Fluidics The purpose of a fluidics system is to transport particles in a fluid stream to the laser beam for interrogation. Three conditions needed for optimal interrogation: •

The stream transporting the particles should pass through the focal point of the laser beam.

•

Optimally one particle should move through the laser beam at one time.

•

Fluidics system needs to be free of air bubbles & debris.

21 | Life Technologies Proprietary & Confidential | 11/7/2011

Fluidics

22 | Life Technologies Proprietary & Confidential | 11/7/2011

23 | Life Technologies Proprietary & Confidential | 11/7/2011

Fluids Attune Focusing Fluid– is an isotonic, buffered, azide-free support/carrier reagent for transporting particles through the capillary assembly to the flow cell for laser interrogation. It contains a preservative and detergent designed to minimize bubble formation. Prevents sample from coming into contact with the walls of the flow cell (optical cuvette). Attune™ Wash Solution – is a ready-to-use solution for removing cellular debris and dyes from the fluidics system of the instrument. Attune™ Shutdown Solution – is a 10X solution that prevents bubble formation in the fluidics system of the instrument. Prepare a 1:10 dilution of the shutdown solution in deionized water. 10% Household Bleach Solution in deionized water (0.5% Sodium hypochlorite) – decontaminates the fluidics lines. Prepare this solution fresh daily and use during the shutdown procedure. Deionized water – used for diluting Attune™ Shutdown Solution and bleach, as well as for long-term storage of the instrument.

24 | Life Technologies Proprietary & Confidential | 11/7/2011

Collection Panel

Run Status indicated the event rate as events per second and the progression of sample collection Collection criteria allows users to define the endpoint of collection Collection controls allows users to run, record, stop sample or clear data Collection mode allows users to run in high sensitivity mode or standard mode as well as choose sample input rate

25 | Life Technologies Proprietary & Confidential | 11/7/2011

Fluidics Functions •

De-bubble is a user-initiated function for clearing bubbles in the fluidics lines of the cytometer.

•

Wash is a user-initiated system cleaning between sticky samples. This function requires user supplied 10% bleach solution.

•

Unclog function is a user-initiated back flush operation to remove clogs from the sample probe and flow cell.

•

Rinse flushes system between samples to minimize carryover. This function is run automatically between samples, but it can also be user initiated.

•

Shutdown is an automated function that decontaminates, cleans, rinses and powers off the cytometer. This mode requires user supplied bleach, Attune™ Wash Solution, and Attune™ Shutdown Fluid.

•

Startup primes the instrument fluidics with Attune™ Focusing Fluid.

•

Stop is used to end any running script.

•

Clear Error is used to erase any error prompts.

26 | Life Technologies Proprietary & Confidential | 11/7/2011

Status Lights

27 | Life Technologies Proprietary & Confidential | 11/7/2011

Routine Maintenance Procedure

Frequency

• Shutdown

Daily

• Visual inspection of sample injection port

Daily

• Visual inspection of fluidics tanks and connections

Daily

• Visual inspection of syringe pumps

Daily

• Computer maintenance

Weekly

• Optical filter cleaning

Monthly

• Fluidics maintenance

As needed*

• Replacing syringes

As needed*

• Changing focusing fluid filter

As needed*

* The frequency of maintenance depends on how often you run the cytometer. If the Attune is to be unused for a period of time exceeding two weeks, perform the shutdown function using distilled water.

28 | Life Technologies Proprietary & Confidential | 11/7/2011

Daily maintenance

29

11/7/2011 | Life Technologies™ Proprietary and confidential

Daily operation - visual inspections • Fluidics compartment: make sure there is no fluid or salt residue on the floor of the compartment, around the connectors, or tube junctions. • Check the fluid levels. Fill/empty as needed focusing fluid wash solution shutdown solution waste • Syringe compartment - make sure there is no fluid or salt residue on the floor of the compartment focusing fluid filter – change if there are any signs of debris/dirt or if the focusing pump stays on for too long. If working correctly, the pump should only be on seconds/less than a minute. syringes – change if they leak or have salt residue build-up

30 | Life Technologies Proprietary & Confidential | 11/7/2011

Daily operation – startup / shutdown • Startup Function Starts the fluidics, optics and electronics • Warms up the lasers (give 15 minutes before running samples) • Initializes the syringe pumps • Primes the instrument fluidics •

• Shutdown Function • Turns off lasers • Cleans and rinses the fluid lines • Refills fluid lines with shutdown solution

• Ensures that: all fluidic lines are clean • Syringe pumps are filled with fresh focusing fluid • Lasers are warmed up to operating temperature •

31 | Life Technologies Proprietary & Confidential | 11/7/2011

• Ensures that: • Fluid lines are filled with a solution that prevents crystal formation and bubbles

Regular Computer Maintenance Procedures

• Back up your experiments on a regular basis to a secondary storage device • Defragment the hard drive of the computer weekly • Minimize memory usage: -When planning the experiments remember to delete parameters that you do not need i.e. only collect the parameters needed

32 | Life Technologies Proprietary & Confidential | 11/7/2011

Questions?

33 | Life Technologies Proprietary & Confidential | 11/7/2011

Optics

Dichroic Mirrors

Band Pass Filters

50mW 405nm Violet Laser 20mW 488 Blue Laser

34 | Life Technologies Proprietary & Confidential | 11/7/2011

Laser Light Scatter Forward Scattered light (FS) is proportional to cell-surface area or size. Side-scattered light (SS) is proportional to cell granularity/internal complexity of the cell. SS is usually collected at 90 degrees to the laser beam.

. Laser

.

. .

.

. . .. .

.

SS detector

35 | Life Technologies Proprietary & Confidential | 11/7/2011

FS detector

Fluorescence- Common Definitions Fluorescence - is the result of a three stage process in certain molecules called fluorophores, or fluorescent dyes.

Absorption spectrum - The wavelength range over which a fluorescent compound can be excited.

Emission spectrum - The range of emitted wavelengths of a fluorescent compound, it is a longer wavelength than the absorption wavelength.

Auto-Fluorescence - Otherwise know as background fluorescence that originates from endogenous sample constituents or from unbound or nonspecifically bound probes.

36 | Life Technologies Proprietary & Confidential | 11/7/2011

This Box is to cover up the word summary and arrow

37 | Life Technologies Proprietary & Confidential | 11/7/2011

Optical Filters

There are five types of optical filters used in flow cytometry: • Bandpass filter (BP) • Longpass filter (LP) • Shortpass filter (SP) • Dichroic mirror (DM) • Neutral density filter (ND) 38 | Life Technologies Proprietary & Confidential | 11/7/2011

Long Pass

Band Pass Filters

Short Pass

Dichroic Mirror

39 | Life Technologies Proprietary & Confidential | 11/7/2011

Product Overview: Attune® Laser Configuration Laser

488 nm Blue 20 mW laser

638 nm Red 50 mW laser

Laser

488 nm Blue 20 mW laser

405 nm Violet 50 mW laser

40 | Life Technologies Proprietary & Confidential | 11/7/2011

Channel BL1 BL2 BL3 BL4 FSC RL1 RL2 SSC

Filter 530/30 574/26 690/50 780/60 488/10 660/20 780/60 638/10

Wavelength Range 551-545 (FITC) 561-587 (PE) 665-715 750-810 483-493 650-670 750-810 633-643

Channel BL1 BL2 BL3 VL1 VL2 VL3 FSC SSC

Filter 530/30 574/26 640LP 450/40 522/31 603/48 405/10 405/10

Wavelength Range 515-545 561-588 >640 430-470 507-537 579-627 400-410 400-410

Compensation •Compensation is the process by which we correct for "spillover“ •Every fluorescent molecule emits light with a particular spectrum unique to that molecule •These emission spectra overlap, in some cases very significantly

530/30 46.8% FITC 0.4% R-PE 0% PerCP 575/26 10.1% FITC 53.9% R-PE 0% PerCP 648 LP 0.1% FITC 8.8% R-PE 99.7% PerCP

41 | Life Technologies Proprietary & Confidential | 11/7/2011

Uncompensated vs. Compensated Data

42 | Life Technologies Proprietary & Confidential | 11/7/2011

Questions?

43 | Life Technologies Proprietary & Confidential | 11/7/2011

Electronics Functions of Electronics: • Converts detected light signals into electronic signals • Electronic signals are process by the computer system • Converts signals from detectors into digital data used for analysis

Electronics and Computers

44 | Life Technologies Proprietary & Confidential | 11/7/2011

Laser

Voltage Pulse in PMT

Voltage

Sample Flow

Laser

Voltage

Time

Time Voltage

Laser

Peak Height Width Area

Time 45 | Life Technologies Proprietary & Confidential | 11/7/2011

Pulse Height

Volts

Electronic Pulse

Pulse Area

Threshold (“trigger”) 0 Pulse Width

Time (µ Seconds)

46 | Life Technologies Proprietary & Confidential | 11/7/2011

SSC

FL2

Electronics: Overview

Time

FL1

FSC

FSC

Time Data Processor

SSC

Time FL1 Time FL2 Time FL3

47 | Life Technologies Proprietary & Confidential | 11/7/2011

Doublet Discrimination

Doublets

Singlets

48 | Life Technologies Proprietary & Confidential | 11/7/2011

Questions?

49 | Life Technologies Proprietary & Confidential | 11/7/2011

Instrument Performance Tracking • Allows you to monitor performance of the instrument • Critical to ensure accuracy and sensitivity of instrument • Includes: •Running the same bead particle set (AttuneTM Performance Beads) •Monitoring changes in the CV •Monitoring changes in mean fluorescent intensity •Tracking linearity of instrument •Evaluating quantum efficiency and background •Sets laser delay •Sets the instrument’s performance baseline • Provides information about all the lasers and detection channels 50 | Life Technologies Proprietary & Confidential | 11/7/2011

Attune Performance Tracking Beads • A mixture of beads of four fluorescence emission intensities in equal concentration − Blank − Dim − Medium − Bright • 3mL vial PN: 4449754

51 | Life Technologies Proprietary & Confidential | 11/7/2011

Running Baseline • Uses performance tracking beads • CSV file obtained from Applied Biosystems® website • Performed any time a new lot of AttuneTM Performance beads are used • Performed after any major maintenance on the instrument • The percent half-peak coefficient of variation (%HPCV) of the brightest bead is recorded • PMT is adjusted to place the brightest bead at target MFI values, and voltage values for each channel are recorded • Relative quantum efficiency (rQ) and relative Background (rB) is calculated for each channel • Linear regression is calculated and recorded • Laser delay setting is also automatically calculated 52 | Life Technologies Proprietary & Confidential | 11/7/2011

53 | Life Technologies Proprietary & Confidential | 11/7/2011

Running Daily Performance Check • Performed after baseline values have been defined • AttuneTM Performance Beads are used to run daily performance measurements to track the performance of the cytometer • Run the performance test at least once per day that the instrument is used • Determines the voltage required to place the brightest bead in the target MFI, and calculates the delta PMT voltages as compared to the baseline. • %HPCV of the bright bead is recorded • Relative quantum efficiency (rQ) and relative Background (rB) is calculated for each channel • Linear regression is calculated and recorded • Laser delay setting is also automatically calculated • Levy-Jennings charts provides a record of %HPCV and PMT voltage to check for shifts and trends

54 | Life Technologies Proprietary & Confidential | 11/7/2011

Daily Performance Check

55 | Life Technologies Proprietary & Confidential | 11/7/2011

Levey-Jennings Reports

56 | Life Technologies Proprietary & Confidential | 11/7/2011

PMT Voltage A calibration curve plot showing limit of detection (LOD), limit of quantification (LOQ), dynamic range, and limit of linearity (LOL).

57 | Life Technologies Proprietary & Confidential | 11/7/2011

What to do if performance check fails 1. 2. 3. 4. 5. 6.

Repeat performance check Check the Performance test troubleshooting section in the users guide. If additional help is required, don’t hesitate to call . Call: 800-831-6844 or 1-800-327-3002, option 4 (5 am-5 pm PST), or email: [email protected] The AB Call Center will open a call or simply transferring the call to the TAC group if assistance is needed. Provide the serial number and contact information www.appliedbiosystems.com/support

Request instrument repair

58 | Life Technologies Proprietary & Confidential | 11/7/2011

Other maintenance

59

11/7/2011 | Life Technologies™ Proprietary and confidential

Other maintenance - Optics Filter holders

• Check for dust or scratches • Remove any dust from the surface with a blower (compressed gas) or a soft brush. • If necessary, gently clean using a clean lens cloth and lens cleaning solution or MeOH • Frequency – monthly or less

60 | Life Technologies Proprietary & Confidential | 11/7/2011

Other maintenance optics • Important notes: Always allow at least 10 min for the lasers to reach operating temp − Powering the instrument on/off within 30 minutes can decrease the laser lifetime. − The “Shutdown” and “Wash scripts” powers off the lasers automatically, allow at least 10 min for the laser to reach operating temperature − If you cancel shutdown, allow at least 10 min for the lasers to reach operating temperature −

61 | Life Technologies Proprietary & Confidential | 11/7/2011

Other maintenance – Fluidics decontamination • Potential problem - contamination − −

Check for cloudiness or debris Discolored solution or brown marks on sensor

• Cleaning instructions: User bulletin # 4468798 Jun 2011 • Frequency: monthly • Perform ‘system flush’ if the instrument will not be used for ≥ 2 weeks

• Replacement part #s - 1L waste - 1L focusing fluid - 250 ml wash soln - 250 ml shutdown soln

62 | Life Technologies Proprietary & Confidential | 11/7/2011

# 90039272 # 90039273 # 90032053 # 90039274

Other maintenance - focusing fluid filter and syringes

Focusing Fluid Filter

10ml Syringe Syringes/Pumps door

63 | Life Technologies Proprietary & Confidential | 11/7/2011

1ml Syringe

Other maintenance – Focusing fluid filter Focusing fluid filter PN 4456564 Crack


The filters will grow some contamination over time. If you can see brownish red spot, and if the filter is intact, it should still keep this growth out of the bottle.
The focusing fluid filter air gap needs to point down ( )
Replacing focusing fluid filter every 6 months reduce the risk of any potential contamination in the lines.
Don’t hesitate to replace focusing fluid filter !

64 | Life Technologies Proprietary & Confidential | 11/7/2011

Other maintenance - Syringes

• Potential problem: Check for leaks − Erratic fluid draws from fluidics tanks or SIP − No fluid draws up from the fluidics tanks or SIP −

• Part numbers: - 1 ml syringe - 10 ml syringe

• Frequency: as needed

65 | Life Technologies Proprietary & Confidential | 11/7/2011

4452079 4452819

Syringe replacement – directions in manual (V 1.2.5) • Run “Shutdown script”. The plunger will be lowered and instrument will turn off • Open the Pump door and remove the plunger lock screw • Unscrew the syringe from the valve by rotating it counter-clockwise • To install a new syringe, pull the plunger down • Align the syringe with valve and syringe plunger holder assembly • Rotate clockwise until the syringe end cap seal hits the bottom of the valve • After bottoming out, rotate clockwise ¼ turn to ensure complete seal • Align the hole in the plunger with the hole in the plunger holder assembly • Insert the plunger lock screw and tighten Notes: • No tools should be used to tighten the syringe to the valve. • Over tightening the syringe beyond the above recommendation could result in damage to the syringe and/or valve. • Proper syringe-to-valve seal is crucial for the operation when fluids are cycling through the system. 66 | Life Technologies Proprietary & Confidential | 11/7/2011

Other maintenace: System flush / long term shutdown • Perform system flush if the Attune will be shutdown for > 2 weeks − 1. Replace all fluidics container (focusing fluid, wash and shutdown tanks) with de-ionized water − 2. Run startup − 3. Run shutdown function using deionized waster on the SIP instead of bleach − 4. When shutdown is complete, empty all fluidics tanks and allow to air dry • This process will prevent crystals from clogging the fluids system.

67 | Life Technologies Proprietary & Confidential | 11/7/2011

Other maintenance: Software • Set up user accounts with operator privileges • Browser − Minimize the number of experiments displayed in the browser − Close all experiments except the one that is currently active (by clicking on the arrow to minimize experiments) − Remove experiments from browser but keep data on hard drive Right click on the experiment name, Click on delete experiment – the following options are displayed > Yes – will delete the experiment and associated files > No – will delete the experiment from the browser and keep the associated files (on the hard drive) > Cancel – will make no changes

68 | Life Technologies Proprietary & Confidential | 11/7/2011

Other maintenance - regular computer maintenance procedures • Back up your experiments on a regular basis to a secondary storage device • Check thumb drives for viruses, run Antivirus software to remove threats • Defragment Hard Drive and run Disk Cleaner (Monthly) • Minimize memory usage: Instrument settings - delete parameters not needed i.e. only collect the parameters needed

69 | Life Technologies Proprietary & Confidential | 11/7/2011

Summary Maintenance Procedure

Frequency

• Start up / Shutdown

Daily

• Visual inspection of sample injection port

Daily

• Visual inspection of fluidics tanks and connections

Daily

• Visual inspection of syringe pumps

Daily

• Computer maintenance

Weekly

• Optical filter cleaning

Monthly

• Fluidics maintenance

Monthly

• Replacing syringes

As needed*

• Changing focusing fluid filter

As needed*

• The frequency of maintenance depends on how often you run the cytometer. • If the Attune is to be un-used for a period of time exceeding two weeks, perform the shutdown function using distilled water.

70 | Life Technologies Proprietary & Confidential | 11/7/2011

Software Overview Main Features: Tracking and Performance Experiment Explorer Collection Panel Ribbons Menus

71 | Life Technologies Proprietary & Confidential | 11/7/2011

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. © 2010 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. NOTICE TO PURCHASER: Limited Use Label License The products shown in this presentation may be covered by one or more Limited Use Label License(s). Please refer to the respective product documentation or the Applied Biosystems website under www.appliedbiosystems.com for the comprehensive license information. By use of these products, the purchaser accepts the terms and conditions of all applicable Limited Use Label Licenses. These products are sold for research use only, and are not intended for human or animal diagnostic or therapeutic uses unless otherwise specifically indicated in the applicable product documentation or the respective Limited Use Label License(s).

72 | Life Technologies Proprietary & Confidential | 11/7/2011