BD Horizon BV421 Annexin V is a sensitive probe for identifying apoptotic cells, binding to negatively charged phospholipid surfaces with a higher affinity for phosphatidylserine (PS) than most other phospholipids. BV421 Annexin V binding is calcium dependent and defined calcium and salt concentrations are required for optimal staining as described in the BV421 Annexin V Staining Protocol. Investigators should note that BV421 Annexin V flow cytometric analysis on adherent cell types (eg, 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 BV421 Annexin V may be used on a cell line (Jurkat).
1. Prepare Camptothecin stock solution (Sigma-Aldrich Cat. No. C-9911): 1 mM in DMSO.
2. Jurkat T cells (ATCC TIB-152).
1. Add Camptothecin (final conc. 4-6 µM) to 1 × 10^6 Jurkat cells.
2. Incubate the cells for 4-6 hr at 37°C.
3. Proceed with the BV421 Annexin V Staining Protocol to measure apoptosis.
1. BD Horizon BV421 Annexin V: 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.
3.10X Annexin Binding Buffer: Not Included. 0.1 M Hepes (pH 7.4) 1.4 M NaCl, 25 mM CaCl2. Store at 4°C. Alternatively, BD Pharmingen™ Annexin V Binding Buffer, 10X concentrate (Cat. No. 556454) may be purchased.
BD Horizon BV421 ANNEXIN V STAINING PROTOCOL
1. Wash cells twice with cold PBS and then resuspend cells in 1× Binding Buffer at a concentration of 1 × 10^6 cells/ml.
2. Transfer 100 µl of the cell suspension (1 × 10^5 cells) to a 5 ml culture tube.
3. Add 5 µl of BV421 Annexin V (for one and two color analysis) and 5 µl of 7-AAD (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 1× Annexin V Binding Buffer to each tube. Analyze by flow cytometry within 1 hr.
SUGGESTED CONTROLS FOR SETTING UP FLOW CYTOMETRY
The following controls are used to set up compensation and quadrants:
1. Unstained cells.
2. Cells stained with BV421 Annexin V alone (no 7-AAD).
3. Cells stained with 7-AAD alone (no BV421 Annexin V).
Other Staining Controls:
A cell line that can be easily induced to undergo apoptosis should be used to obtain positive control staining with BV421 Annexin V alone or with BV421 Annexin V and 7-AAD. It is important to note that the basal level of apoptosis and necrosis varies considerably within a population. Thus, even in the absence of induced apoptosis, most cell populations will contain a minor percentage of cells that are positive for apoptosis (BV421 Annexin V positive, 7-AAD negative or BV421 Annexin V positive, 7-AAD positive).
The untreated population is used to define the basal level of apoptotic and dead cells. The percentage of cells that have been induced to undergo apoptosis is then determined by subtracting the percentage of apoptotic cells in the untreated population from the percentage of apoptotic cells in the treated population. Since cell death is the eventual outcome of cells undergoing apoptosis, cells in the late stages of apoptosis will have a damaged membrane and stain positive for 7-AAD as well as for BV421 Annexin V. Thus, the assay does not distinguish between cells that have already undergone an apoptotic cell death and those that have died as a result of necrotic pathway, because in either case the dead cells will stain with both BV421 Annexin V and 7-AAD.