Cy™5 Annexin V is a sensitive probe for identifying apoptotic cells, binding to negatively charged phospholipid surfaces (Kd of ~5 x 10e2) with a higher affinity for phosphatidylserine (PS) than most other phospholipids. Cy™5 Annexin V binding is calcium dependent and defined calcium and salt concentrations are required for optimal staining as described in the Cy™5 Annexin V Staining Protocol. Investigators should note that Cy™5 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 Cy™5 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 10e6 Jurkat cells.
2. Incubate the cells for 4-6 hr at 37°C.
3. Proceed with the Cy™5 Annexin V Staining Protocol to measure apoptosis.
Cy™5 ANNEXIN V STAINING PROTOCOL
1. Cy™5 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 Binding Buffer: Not Included. 0.1 M Hepes (pH 7.4) 1.4 M NaCl, 25 mM CaCl2. Store at 4°C. Alternatively, Annexin V Binding Buffer, 10X concentrate (Cat. No. 556454) may be purchased.
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 Cy™5 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 1X Binding Buffer to each tube. Analyze by flow cytometry within 1 hr.
SUGGESTED CONTROLS FOR SETTING UP FLOW CYTOMETRY
Cy™5 emission wavelength is 670 nm and excitation is 625-650 nm. Cy5 is optimized for FL4 fluorescence on the BD FACSCalibur™ flow cytometer. For the BD FACSVantage™ flow cytometer, the recommended filter for emission is 675/20.
The following controls are used to set up compensation and quadrants:
1. Unstained cells.
2. Cells stained with Cy™5 Annexin V alone (no 7-AAD).
3. Cells stained with 7-AAD alone (no Cy™5 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 Cy™5 Annexin V and/or Cy™5 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 (Cy™5 Annexin V positive, 7-AAD negative or Cy™5 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 Cy™5 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 Cy™5 Annexin V and 7-AAD.