Resuspend cells in cold 1X binding buffer to a concentration of 1 x 107 cells/mL.
Add 100 uL of cells (1 x 106) to each labeled tube.
Add 10 uL of Annexin V-FITC to appropriate tubes.
Gently vortex each tube and incubate for 15 minutes on ice, protected from light.
Without washing, add 300 uL of cold 1X binding buffer to each tube.
Analyze by flow cytometry by gating on viable cells by light scatter.
Flow Cytometry: 10 ul/10^6 cells
A number of changes in cell structure occur during apoptosis. One major change is the loss of membrane phospholipid asymmetry, with translocation of phosphatidylserine (PS) from the inner leaflet of the phospholipid bilayer to the external surface.1 Annexin V (Mr 36-kDa), a member of the annexin family of calcium-dependent phospholipid binding proteins, has a high affinity for phosphatidylserine (PS)-containing phospholipid bilayers.2,3. When PS is exposed on the extracellular face of a cell membrane, Annexin V binds with an affinity of approximately 7 nM4. Fluorochrome-conjugated Annexin V can be used to monitor changes in cell membrane phospholipid asymmetry,5-8 thereby providing a convenient tool for detection of apoptotic cells.
Annexin V-FITC is a labeled Annexin V that can be used to evaluate subpopulations of cells along the apoptotic pathway without counterstaining with propidium iodide or other DNA dyes. Analysis is performed on a flow cytometer equipped with a 488 nm argon laser and gated on viable cells by light scatter (see flow histograms below). Alternatively, the cells can be examined by fluorescence microscopy.
2. Tait, J.F., et al. 1989. J. Biol. Chem. 264:7944.
3. Andree, H.A., et al. 1990. J. Biol. Chem. 265:4923.
4. Thiagarajan, P., et al. 1990. J. Biol. Chem. 265:17420.
5. Dachary, P.J., et al. 1993. Blood 81:2554.
6. Koopman, G., et al. 1994. Blood 84:1415.
7. Martin, S.J., et al. 1995. J. Exp. Med. 182:1545.
8. Wood, B.L., et al. 1996. Blood 88:1873
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Product Notes
The Aposcreen Annexin V (Catalog #AAA670381) is an Antibody and is intended for research purposes only. The product is available for immediate purchase. AAA Biotech's Aposcreen Annexin V can be used in a range of immunoassay formats including, but not limited to, Flow Cytometry, Fluorescence Microscopy. Suggested Staining Protocol: Wash cells twice in cold PBS; remove the PBS from the cell pellet after each wash. Resuspend cells in cold 1X binding buffer to a concentration of 1 x 107 cells/mL. Add 100 uL of cells (1 x 106) to each labeled tube. Add 10 uL of Annexin V-FITC to appropriate tubes. Gently vortex each tube and incubate for 15 minutes on ice, protected from light. Without washing, add 300 uL of cold 1X binding buffer to each tube. Analyze by flow cytometry by gating on viable cells by light scatter. Flow Cytometry: 10 ul/10^6 cells. Researchers should empirically determine the suitability of the Aposcreen Annexin V for an application not listed in the data sheet. Researchers commonly develop new applications and it is an integral, important part of the investigative research process. It is sometimes possible for the material contained within the vial of "Aposcreen Annexin V, Antibody" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
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