BD, 561501, BD Horizon™ V500 Annexin V

Reactivity: Human (QC Testing)
Application: Flow cytometry (Routinely Tested)
Vol. Per Test: 5 µl
RRID: AB_10694254
Storage Buffer: Aqueous buffered solution containing BSA, protein stabilizer, and ≤0.09% sodium azide.
Regulatory Status: RUO
Preparation And Storage: Preparation And Storage Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with BD Horizon™ V500 under optimum conditions, and unreacted BD Horizon™ V500 was removed.
Recommended Assay Procedures: Recommended Assay Procedures BD™ Horizon V500 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. V500 Annexin V binding is calcium-dependent. Therefore, defined calcium and salt concentrations are required for optimal staining as described in the V500 Annexin V Staining Protocol. Investigators should note that V500 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 of how V500 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 × 10^6 Jurkat cells. 2. Incubate the cells for 4-6 hr at 37°C. 3. Proceed with the V500 Annexin V Staining Protocol to measure apoptosis. V500 ANNEXIN V STAINING PROTOCOL Reagents 1. V500 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. Staining 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 V500 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× 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 V500 Annexin V alone (no 7-AAD). 3. Cells stained with 7-AAD alone (no V500 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 V500 Annexin V and/or V500 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 (V500 Annexin V positive, 7-AAD negative or V500 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 V500 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 V500 Annexin V and 7-AAD.
Product Notices: Product Notices This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test). Source of all serum proteins is from USDA inspected abattoirs located in the United States. 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. BD Horizon V500 has a maximum absorption of 415 nm and maximum emission of 500 nm. Before staining with this reagent, please confirm that your flow cytometer is capable of exciting the fluorochrome and discriminating the resulting fluorescence. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.