Aposcreen Annexin V Protein : Biotin (OPSB00050)

Catalog No: OPSB00050

Size:100Tests

Amount Used: 10 uL/106 cells Two-color flow cytometry analysis of untreated (left panel) and irradiated (right panel) BALB/c thymocytes stained with annexin V-FITC and propidium iodide. Early apoptotic cells are located in the lower right quadrant of the two displays.

Product Info

• Host: Expressed in E. coli
• Conjugation: Biotin
• Application: FC, IHC, ICC
• Additional Information: Description: ApoScreen Annexin V is a recombinant protein that is expressed in E. coli. 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. Annexin V, a member of the annexin family of calcium-dependent phospholipid binding proteins, has a high affinity for phosphatidylserine (PS)-containing phospholipid bilayers. Conjugated Annexin V can be used to monitor changes in cell membrane phospholipid asymmetry, thereby providing a convenient tool for detection of apoptotic cells.
  Suggested Staining Protocol:
  1. Wash cells twice in cold PBS; remove the PBS from the cell pellet after the second wash.
  2. Resuspend cells in cold binding buffer (e.g. 10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl2, 0.1% BSA) to a concentration of 1 x 106 to 1 x 107 cells/mL.
  3. Add 100 uL of cells (1 x 105 to 1 x 106) to each of four labeled tubes.
  4. Add 10 uL of labeled annexin V to tube #2 and tube #4.
  5. Gently vortex each tube and incubate for 15 minutes on ice, protected from light.
  6. Without washing, add 380 uL of cold 1X binding buffer to each tube. Add 10 uL of PI (from a 50 ug/ml stock solution) to tube #3 and tube #4.
  7. Immediately analyze by flow cytometry. Note: the 4-tube protocol above is for initially adjusting settings on the flow cytometer; subsequent assays should basically require the protocol described for 'tube #4,' i.e. cells stained with both labeled annexin V and PI.
  HANDLING AND STORAGE
  The Biotin (BIOT) conjugate is supplied as 100 tests in 1.0 mL of 1X Binding Buffer. Store at 2-8C.
• Reconstitution and Storage: Store at 2-8C
• Dilution: Flow Cytometry: Biotin conjugate
  10 ul/10⁶ cells
• Concentration: Lot specific
• Characterization: To insure acceptable performance, each batch of product is tested by flow cytometry to conform to thecharacteristics of a standard reference reagent.
• Storage: Annexin V- BIOT: Store at 2- 8 C
  
• Application Info: Blocking Annexin V-conjugates, Flow cytometry, Fluorescence microscopy
• Other Applications Data: Since applications vary, you should determine the optimum workingamount of the product that is appropriate for your specific need.
• Reference: 1. Fadok, V.A. et al. 1992. J. Immunol. 148:22072. 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.
• Warning: Reagents contain sodium azide. Sodium azide is very toxic if ingested or inhaled. Avoid contact with skin,eyes, or clothing. Wear eye or face protection when handling. If skin or eye contact occurs, wash withcopious amounts of water. If ingested or inhaled, contact a physician immediately. Sodium azide yields toxichydrazoic acid under acidic conditions. Dilute azide- containing compounds in running water before discardingto avoid accumulation of potentially explosive deposits in lead or copper plumbing.