Description
Annexin V is a sensitive probe for identifying apoptotic cells, binding to negatively charged phospholipid surfaces (Kd of ~5 x 10e-2) with a higher affinity for phosphatidylserine (PS) than most other phospholipids. Annexin V binding is calcium dependent and defined calcium and salt concentrations are required for optimal staining as described in the Annexin V Staining Protocol. Investigators should note that Annexin V flow cytometric analysis on adherent cell types (e.g HeLa, NIH 3T3, etc.) is not routinely tested as specific membrane damage may occur during cell detachment or harvesting. Methods for utilizing Annexin V for flow cytometry on adherent cell types, however, have been previously reported (Casiola-Rosen et al. and van Engelend et al.). INDUCTION OF APOPTOSIS BY CAMPTOTHECIN The following protocol is provided as an illustration on how Annexin V may be used on a cell line (Jurkat). Materials 1. Prepare Camptothecin stock solution (Sigma-Aldrich Cat. No. C-9911): 1 mM in DMSO. 2. Jurkat T cells (ATCC TIB-152). Procedure 1. Add Camptothecin (final conc. 4-6 µM) to 1 x 10e6 Jurkat cells. 2. Incubate the cells for 4-6 hr at 37°C. 3. Proceed with the Annexin V Staining Protocol to measure apoptosis. INDUCTION OF APOPTOSIS USING AN ANTI-HUMAN CD95 (FAS) ANTIBODY The following protocol is provided as an illustration on how Annexin V may be used on a human cell line. Materials 1. A cell line or primary cells that can easily be induced to undergo apoptosis by human Fas mAb. Examples include Daudi lymphoma cells (ATCC CCL-213) and Jurkat T cells (ATCC TIB-152). It is important to note that there can be significant variation between cell lines regarding the level of apoptosis that can be induced through the Fas receptor. Also, not all cell types which express the Fas antigen will necessarily undergo Fas-mediated apoptosis. The cell lines mentioned above are good positive controls as they are strongly induced to undergo apoptosis by Fas mAb. 2. Anti-human CD95 (Fas) mAb, clone DX2 (Cat. No. 555670). 3. Recombinant Protein G (Sigma-Aldrich cat.no. P4689). We have found that the addition of Protein G to the tissue culture medium can significantly enhance the efficiency of the DX2 clone to induce apoptosis. 4. T25 tissue culture flasks. 5. IMDM or RPMI 1640 medium with 10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine and 1% antibiotics (penicillin/ streptomycin; 100 U/ml). This supplemented medium is simply referred to as 'medium' below. Procedure 1. Maintain the cells in culture and change the medium one day before inducing apoptosis. 2. Induction of apoptosis: Add 0.5 - 2 µg/ml of the anti-CD95 antibody (DX2 clone) and 1-2 µg/ml Protein G to a T25 flask with medium containing ~0.5 × 10e6 cells/ml. Negative controls should consist of: (a) ~0.5 × 10e6 cells/ml with medium alone (no mAb or Protein G), and (b) ~0.5 × 10e6 cells/ml with medium and 1 µg/ml Protein G alone (no mAb). 3. Incubate the cells for 2 to 12 hr at 37°C 4. Proceed with the Annexin V Staining Protocol to measure apoptosis. Apoptosis can also be observed by light microscopy, gel electrophoresis (DNA fragmentation ladders) or by using a DNA fragmentation-based flow cytometry assay system such as the APO-BRDU™ Kit (Cat. No. 556405) or the APO-DIRECT™ Kit (Cat. No. 556381). ANNEXIN V STAINING & BLOCKING PROTOCOL Reagents 1. Biotin (Cat.No. 556418), FITC (Cat.No. 556420), PE (Cat.No. 556422), APC (Cat.No. 550474), Cy5 (Cat.No. 559933), or Cy5.5 (Cat.No. 559935) conjugated Annexin V reagents: Not Included. Use 5 µl per test. 2. 7-Amino-Actinomycin D (7-AAD): Not included. 7-AAD (Cat.No. 559925) is a convenient, ready-to-use nucleic acid dye with fluorescence detectable in the far red range of the spectrum. Use 5 µl per test. 3. Propidium Iodide (PI): Not Included. PI (cat.no. 556463) is a convenient, ready-to-use nucleic acid dye. Use up to 10 µl per test of a 50 µg/ml solution. The optimal concentration of PI may vary among cell lines where 10 µl of a 50 µg/ml stock is most likely the maximum to be required. Less may yield optimal results in some experimental systems. 4. 10X Binding Buffer: Not Included. 0.1 M Hepes (pH 7.4) 1.4 M NaCl, 25 mM CaCl2. Store at 4°C. Alternatively, catalog number 556454 may be purchased. 5. Purified Recombinant Annexin V (Cat.No. 556416): Included. Staining 1. Wash cells twice with cold PBS and then resuspend cells in 1X Binding Buffer at a concentration of 1 x 10e6 cells/ml. 2. Transfer 100 µl of the solution (1 x 10e5 cells) to a 5 ml culture tube. 3. Add 5 µl of fluorochrome conjugated-Annexin V (for one and two color analysis) and/or 5 µl of 7-AAD or 10 µl PI (for two color analysis only). 4. Gently vortex the cells and incubate for 15 min at RT (25°C) in the dark. 5. Add 400 µl of 1X Binding Buffer to each tube. Analyze by flow cytometry within 1 hr. Blocking 1. Wash cells twice with cold PBS and then resuspend cells in 1X Binding Buffer at a concentration of 1 x 10e6 cells/ml. 2. Transfer 100 µl of the solution (1 x 10e5 cells) to a 5 ml culture tube. 3. Add 5-15 µg of purified recombinant Annexin V. The amount of purified recombinant Annexin V required to saturate binding sites may vary according to cell type and stage of apoptosis. In some cases, investigators may need to reduce the number of cells to 0.5 x 10e5 and still add 5-15 µg of recombinant Annexin V to obtain optimal results. Titration is strongly recommended. 4. Gently vortex the cells and incubate for 15 min at room temperature. 5. Add 5 µl of fluorochrome conjugated-Annexin V (for one and two color analysis) and/or 5 µl of 7-AAD or 10 µl PI (for two color analysis only). 6. Gently vortex the cells and incubate for 15 min at room temperature in the dark. 7. Add 400 µl of 1X Binding Buffer to each tube. Analyze by flow cytometry as soon as possible (within 1 hr).
Store product at -80°C prior to use or for long term storage of stock solutions. Avoid multiple freeze-thaws of product.
1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
3. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
4. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
