User Protocol PF032 Rev. 23 May 2005 RFH Page 1 of 8

Annexin V FITC Apoptosis Detection Kit Cat. No. PF032

Table of Contents

Page

Storage of Kit Components Intended Use Background Principle of the Assay Materials Provided Materials Required but not Provided Precautions and Recommendations General Procedural Notes Detailed Protocol a. RAPID Annexin Binding b. Conventional Annexin Binding c. Annexin Binding with Adherent Cells d. Analysis by Flow Cytometry e. Analysis by Fluorescence Microscopy Evaluation of Results Validation References Reagent Stability

1 1 1 2 2 3 3 3 3 3 4 4 4 4 5 6 8 8

Storage of Annexin V-FITC Kit Components Annexin V-FITC kit components are shipped on gel ice pack. Upon receipt, store kit at 2-8oC. To avoid reagent loss in tube caps, briefly pulse spin all small tubes before removing caps. See Precautions and Recommendations. Intended Use The Calbiochem Annexin V-FITC Apoptosis Detection Kit is a non-isotopic system for the identification of cell membrane alterations that accompany programmed cell death. Detection may be performed by flow cytometry or by fluorescence microscopy. Background Apoptosis is a fundamental mode of cell death which performs a regulatory function during normal development, in tissue homeostasis, and in some disease processes. In normal viable cells phosphatidyl serine (PS) is located on the cytoplasmic surface of the cell membrane. Upon induction of apoptosis, rapid alterations in the organization of phospholipids in most cell types occurs leading to exposure of PS on the cell surface. Recognition of PS by phagocytes in vivo results in the removal of cells programmed to die thus apoptosis is not commonly associated with the local inflammatory response which accompanies necrosis.

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User Protocol PF032 Rev. 23 May 2005 RFH Page 2 of 8

In vitro detection of externalized PS can be achieved through interaction with the anticoagulant annexin V. In the presence of calcium, rapid high affinity binding of annexin V to PS occurs. PS translocation to the cell surface precedes nuclear breakdown, DNA fragmentation, and the appearance of most apoptosis-associated molecules making annexin V binding a marker of early-stage apoptosis.

Annexin V Ca++

Principle of the Assay In this assay a fluorescein isothiocyanate (FITC) conjugate of Annexin V is used allowing detection of apoptosis by flow cytometry or by fluorescence microscopy. Since membrane permeabilization is observed in necrosis, necrotic cells will also bind Annexin V-FITC. Propidium iodide is used to distinguish between viable, early apoptotic, and necrotic or late apoptotic cells. Necrotic cells will bind Annexin V-FITC and stain with propidium iodide while propidium iodide will be excluded from viable (FITC negative) and early apoptotic (FITC positive) cells. In the absence of phagocytosis final stages of apoptosis involve necrotic-like disintegration of the total cell, thus cells in late apoptosis will be labeled with both FITC and propidium iodide. A RAPID protocol has been developed for Annexin V-FITC binding directly in tissue culture media (see page 4). This obviates the need for tedious centrifugation and wash steps which increase the occurrence of mechanical membrane disruption. In addition, since apoptosis is a dynamic process that is ongoing once cells are removed from culture conditions and continues throughout experimental processing, the RAPID protocol is recommended for the detection of cells in early apoptosis. Materials Provided Note: The Annexin V-FITC Apoptosis Detection Kit supplies sufficient reagents for 100 tests. • ANNEXIN V-FITC: 200 µg/ml recombinant Annexin V conjugated to fluorescein isothiocyanate (FITC) • 5X BINDING BUFFER: • MEDIA BINDING REAGENT: A reagent designed to enhance binding of Annexin V to PS in tissue culture media • PROPIDIUM IODIDE: Supplied at 30 µg/ml Materials Required But Not Provided

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User Protocol PF032 Rev. 23 May 2005 RFH Page 3 of 8

• • • • • • • • •

PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4-7H2O, 1.4 mM KH2PO4, adjust pH to 7.4) 2-20 µl, 20-200 µl, and 200-1000 µl precision pipetters with disposable tips Microcentrifuge tubes Adjustable speed microcentrifuge Glass microscope slides (for microscope analysis only) Glass coverslips (for microscope analysis only) dH2O Ice Fluorescence microscope or flow cytometer

Precautions and Recommendations 1. For optimal results READ THESE INSTRUCTIONS BEFORE USING THIS KIT. 2. To avoid reagent loss in tube caps, briefly pulse spin all tubes before removing caps. 3. Propidium iodide may be harmful by ingestion or absorption through skin and may cause irritation to the eyes. It should be treated as a possible mutagen. 4. Gloves, lab coat, and protective eyewear should be worn. General Procedural Notes 1. Apoptosis detection with the Annexin V-FITC kit should be performed on live cells. Fixation procedures may interfere with the observation of the FITC signal. It may be possible to fix cells following labeling, however excess calcium should be provided to prevent the reversal of the Annexin-PS interaction. 2. Analysis should be performed immediately after binding of Annexin V-FITC since apoptosis will be an ongoing process. Loss of FITC signal may occur after one hour. 3. Propidium iodide and Annexin V-FITC are light sensitive. Minimize exposure to light. Use a covered ice bucket or aluminum foil where necessary. 4. Successful detection of early apoptosis with Annexin V-FITC is dependent upon several factors including cell type and level of membrane PS, amount of PS that is exposed during apoptosis, method (or agent) inducing apoptosis, time of apoptotic induction, as well as degree of mechanical manipulation. It may be necessary to optimize these procedures for your particular application. Detailed Protocol Three protocols are provided. One for the binding of Annexin V-FITC directly in tissue culture media (RAPID Annexin V Binding), one for the binding of Annexin V-FITC in binding buffer (Conventional Annexin V Binding) following a PBS wash, and another for binding of Annexin V-FITC with adherent cells. The RAPID protocol is recommended where possible. For all protocols prepare enough 1X Binding Buffer (0.5 ml/sample for RAPID binding and 1.0 ml/sample for Conventional binding) by diluting the 5X Binding Buffer concentrate 1:5 with dH2O. Place on ice. RAPID Annexin V Binding 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Adjust the cell suspension concentration to approximately 1 x 106 cells/ml. Transfer 0.5 ml of cell suspension from tissue culture flask (5 x 105 cells) to a microfuge tube. Add 10 µl Media Binding Reagent. Add 1.25 µl Annexin V-FITC. Incubate 15 minutes at room temperature (18 – 24 oC) in the dark. Centrifuge at 1000 x g for 5 minutes at room temperature. Remove media. Gently resuspend cells in 0.5 ml cold 1X Binding Buffer. Add 10 µl Propidium Iodide. Place samples on ice and away from light. Analyze by flow cytometry or fluorescence microscopy immediately.

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User Protocol PF032 Rev. 23 May 2005 RFH Page 4 of 8

Conventional Annexin V Binding 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Adjust the cell suspension concentration to approximately 1 x 106 cells/ml. Transfer 0.5 ml of cell suspension from tissue culture flask (5 x 105 cells) to a microfuge tube. Centrifuge at 1000 x g for 5 minutes at room temperature. Remove media. Gently resuspend cells in 0.5 ml cold PBS. Centrifuge at 1000 x g for 5 minutes at room temperature. Remove PBS. Gently resuspend cells in 0.5 ml cold 1X Binding Buffer. Add 1.25 µl Annexin V-FITC. Incubate 15 minutes at room temperature (18 – 24 oC) in the dark. Centrifuge at 1000 x g for 5 minutes at room temperature. Remove supernatant. Gently resuspend cells in 0.5 ml cold 1X Binding Buffer. Add 10 µl Propidium Iodide. Place samples on ice and away from light. Analyze by flow cytometry or fluorescence microscopy immediately.

Annexin V Binding with Adherent Cells 1. Transfer media from flask of adherent cells to a 15 ml conical tube and place on ice. Note: This media will contain cells that have become detached from the flask during the cell death process. 2. Gently wash cells in flask with 10 ml PBS. Remove PBS. 3. Add 1-2 ml 0.5X trypsin and incubate just until cells appear detached by microscopic evaluation. 4. Release cells from flask with firm tapping. 5. Gently resuspend cells in media from step 1 OR 1X cold binding buffer to approximately 1 x 106 cells/ml. 6. Transfer 0.5 ml of cell suspension to a microfuge tube and continue as described in the RAPID Protocol steps 3 – 10 OR the Conventional Annexin V Binding Protocol steps 7 - 13. Analysis by Flow Cytometry A flow cytometer emitting an excitation light at 488 nm from an argon ion laser should be used to quantify the Annexin V-FITC and propidium iodide signals. To set up the flow cytometer use apoptosis-induced cells stained with FITC only and apoptosis-induced cells labeled with only propidium iodide. The FITC signal can be detected by FL1 (FITC detector) at 518 nm. Propidium iodide fluoresces at 620 nm and can be detected by FL2 (the phycoerythrin fluorescence detector). Perform necessary adjustments to minimize overlap between these two measurements. The log of annexin V-FITC fluorescence should be displayed on the X axis and the log of propidium iodide fluorescence; on the Y axis of the data report. The flow cytometer may also be programmed to display additional parameters such as forward and side (90o) scatter. Analysis by Fluorescence Microscopy Transfer 25 – 50 µl of cell suspension following addition of propidium iodide onto a glass microscope slide. Cover with a glass coverslip and view immediately using a fluorescence microscope equipped with FITC and rhodamine filter sets. Addition of mounting media to the sample will dilute the calcium concentration and may result in the reversal of Annexin V-FITC binding.

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User Protocol PF032 Rev. 23 May 2005 RFH Page 5 of 8

Evaluation of results Flow Cytometry

A sample cytogram is shown above. Viable cells do not bind Annexin V-FITC or propidium iodide as reflected in the lower left-hand quadrant of the dot plot. Early apoptotic cells with exposed PS but intact cell membranes bind Annexin V-FITC but exclude propidium iodide. Fluorescence from this population is reported in the lower righthand quadrant. Necrotic or apoptotic cells in terminal stages will be both Annexin V-FITC and propidium iodide positive and will be reported in the upper right-hand quadrant. A small percentage of normal cell death should be expected in routine cultures of untreated cells. Alterations in light scattering properties as measured by flow cytometry can also be used to monitor cells undergoing apoptosis (8, 9, and 10). For example, untreated Jurkat cells have high forward scatter. Upon induction of apoptosis Jurkat cell shrinkage and membrane blebbing occurs and a marked increase in side scatter is observed. A combination of changes in light scatter and Annexin V-FITC binding may be used to confirm or support conclusions drawn from measurements of a single parameter. Fluorescence Microscopy Using the FITC filter (blue light) on a fluorescence microscope positive Annexin V-FITC staining will appear bright apple green on the cell membrane surface. Using the rhodamine filter (green light) cells that are positive for propidium iodide will appear with various intensities of yellow-red throughout the cytoplasm. Early apoptotic cells will be unstained with propidium iodide or show background fluorescence, while necrotic and late apoptotic cells will have yellow-red cytoplasm and red nuclear staining with green fluorescence surrounding the perimeter of the cell. Membrane blebbing and cell shrinkage may be observable with late apoptotic cells.

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User Protocol PF032 Rev. 23 May 2005 RFH Page 6 of 8

Validation RAPID Annexin V-FITC binding is identical to results with Conventional Protocol With Apoptotic Jurkat Cells. Jurkat cells were incubated with 0.5 ug/ml actinomycin D for either 4.5 (Middle) or 19 (Bottom) hours. Untreated shown in the TOP panel of each section. Annexin V-FITC was then bound in either binding buffer (using the Conventional protocol) or directly in either RPMI, or DMEM or McCoy’s 5A (RAPID Protocol). The log of both PI and FITC fluorescence is reported.

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User Protocol PF032 Rev. 23 May 2005 RFH Page 7 of 8

RAPID Annexin V-FITC binding is identical to results with Conventional Protocol With Apoptotic HL60 Cells. Apoptosis was induced by 30 minutes of UV irradiation followed by either 4 (Middle) or 18 (Bottom) hours incubation at 37°C. Untreated cells are shown in the Top panel of each section. Annexin V-FITC was then bound in either binding buffer (using the Conventional protocol) or directly in either RPMI, or DMEM or McCoy’s 5A (RAPID Protocol).

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User Protocol PF032 Rev. 23 May 2005 RFH Page 8 of 8

References Darzynkiewicz, Z., et al. 1997. Cytometry 27: 1-20. Frey, T. 1997. Cytometry 28: 253-263. Wyllie, A.H.,1993. Br. J. Cancer 67: 205-208. Boersma, A.W.M., et al. 1996. Cytometry 24: 123-130. White, E. 1996. Genes and Devel. 10: 1-15. Martin, S.J., et al. 1995. J. Exp. Med. 182: 1545-1556. Koopman, G., et al. 1994. Blood 84: 1415-1420. Darzynkiewicz, Z., et al. 1992. Cytometry 13: 795-808. Fadok, V.A., et al. 1992. J. Immunology 148: 2207-2216. Kerr, J.F.R., et al. 1972. Cancer 26: 239-257. Reagent Stability The Annexin V-FITC kit is stable until the expiration date stated on the package label. Reagents should not be used beyond the expiration date.

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