Description
APC 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. APC Annexin V binding is calcium dependent and defined calcium and salt concentrations are required for optimal staining as described in the APC Annexin V Staining Protocol. Investigators should note that APC 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 APC 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 APC Annexin V Staining Protocol to measure apoptosis. APC ANNEXIN V STAINING PROTOCOL Reagents 1. APC 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, catalog number 556454 may be purchased. 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 APC 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 The following controls are used to set up compensation and quadrants: 1. Unstained cells. 2. Cells stained with APC Annexin V alone (no 7-AAD). 3. Cells stained with 7-AAD alone (no APC 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 APC Annexin V and/or APC 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 (APC Annexin V positive, 7-AAD negative or APC 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 APC 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 APC Annexin V and 7-AAD.
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
3. 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.
4. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
5. This APC-conjugated reagent can be used in any flow cytometer equipped with a dye, HeNe, or red diode laser.
6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
