V450 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. V450 Annexin V binding is calcium-dependent and defined calcium and salt concentrations are required for optimal staining as described in the V450 Annexin V Staining Protocol. Investigators should note that V450 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 V450 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 V450 Annexin V Staining Protocol to measure apoptosis.
V450 ANNEXIN V STAINING PROTOCOL
V450 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. 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. Propidium Iodide (PI) is a standard flow cytometric viability probe and is used to distinguish viable from nonviable cells.
1. V450 Annexin V: Included. Use 5 µl per test.
2. 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.
3. 10× 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.
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 solution (1 × 10^5 cells) to a 5 ml culture tube.
3. Add 5 µl of V450 Annexin V.
4. Add 10 µl PI. 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.
5. Gently vortex the cells and incubate for 15 min at RT (25 °C) in the dark.
6. Add 400 µl of 1× 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 V450 Annexin V (no PI).
3. Cells stained with PI (no V450 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 V450 Annexin V and/or V450 Annexin V and PI. 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 (V450 Annexin V positive, PI negative or V450 Annexin V positive, PI 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 PI as well as for V450 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 V450 Annexin V and PI.