BD Horizon™ BV711 Annexin V 563972

Reactivity: Human (QC Testing)
Isotype: null null
Application: Flow cytometry (Routinely Tested)
Vol. Per Test: 5 µl
RRID: AB_2869537
Storage Buffer: Aqueous buffered solution containing BSA 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.
Recommended Assay Procedures: Recommended Assay Procedures BD Horizon BV711 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. BV711 Annexin V binding is calcium dependent and defined calcium and salt concentrations are required for optimal staining as described in the BV711 Annexin V Staining Protocol. Investigators should note that BV711 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 on how BV711 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 BV711 Annexin V Staining Protocol to measure apoptosis. Reagents 1. BD Horizon BV711 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. BD Horizon BV711 ANNEXIN V STAINING PROTOCOL 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 cell suspension (1 × 10^5 cells) to a 5 ml culture tube. 3. Add 5 µl of BV711 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× Annexin V 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 BV711 Annexin V alone (no 7-AAD). 3. Cells stained with 7-AAD alone (no BV711 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 BV711 Annexin V alone or with BV711 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 (BV711 Annexin V positive, 7-AAD negative or BV711 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 BV711 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 BV711 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). 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. Source of all serum proteins is from USDA inspected abattoirs located in the United States. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors. BD Horizon Brilliant Violet 711 is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,227,187; 8,455,613; 8,575,303; 8,354,239. BD Horizon Brilliant Stain Buffer is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,575,303; 8,354,239. Cy is a trademark of GE Healthcare. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.