PE Annexin V is a sensitive probe for identifying apoptotic cells, binding to negatively charged phospholipid surfaces (Kd of ~5 x 10^-2) with a higher affinity for phosphatidylserine (PS) than most other phospholipids. PE Annexin V binding is calcium dependent and defined calcium and salt concentrations are required for optimal staining as described in the PE Annexin V Staining Protocol. Investigators should note that PE 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 PE 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 x 10^6 Jurkat cells.
2. Incubate the cells for 4-6 hr at 37°C.
3. Proceed with the PE Annexin V Staining Protocol to measure apoptosis.
PE ANNEXIN V STAINING PROTOCOL
PE Annexin V is used to quantitatively determine the percentage of cells within a population that are actively undergoing apoptosis. It relies on the property of cells to lose membrane asymmetry in the early phases of apoptosis. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner leaflet of the plasma membrane to the outer leaflet, thereby exposing PS to the external environment. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for PS, and is useful for identifying apoptotic cells with exposed PS. 7-Amino-Actinomycin (7-AAD) is a standard flow cytometric viability probe and is used to distinguish viable from nonviable cells. Viable cells with intact membranes exclude 7-AAD, whereas the membranes of dead and damaged cells are permeable to 7-AAD. Cells that stain positive for PE Annexin V and negative for 7-AAD are undergoing apoptosis. Cells that stain positive for both PE Annexin V and 7-AAD are either in the end stage of apoptosis, are undergoing necrosis, or are already dead. Cells that stain negative for both PE Annexin V and 7-AAD are alive and not undergoing measurable apoptosis.
1. PE Annexin V (component no. 51-65875X): Use 5 µl per test.
2. 7-Amino-Actinomycin (7-AAD) (component no. 51-68981E) is a convenient, ready-to-use nucleic acid dye. Use 5 µl per test.
3. 10X Annexin V Binding Buffer (component no. 51-66121E): 0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl2. For a 1X working solution, dilute 1 part of the 10X Annexin V Binding Buffer to 9 parts of distilled water.
1. Wash cells twice with cold PBS and then resuspend cells in 1X Binding Buffer at a concentration of 1 x 10^6 cells/ml.
2. Transfer 100 µl of the solution (1 x 10^5 cells) to a 5 ml culture tube.
3. Add 5 µl of PE Annexin V and 5 µl 7-AAD.
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.
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 PE Annexin V (no 7-AAD).
3. Cells stained with 7-AAD (no PE 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 PE Annexin V and/or PE 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 (PE Annexin V positive, 7-AAD negative or PE 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 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 PE 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 PE Annexin V and 7-AAD.