Annexin V-Cy5 Apoptosis Kit

Details for Product No. ABIN411983
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Target Name (Antigen)
Synonyms anx, anx5, ANX V, anxa5, cb989, wu:fa98f06, wu:fj10f10, MGC89158, ANX5, ENX2, PP4, RPRGL3, Anx5, R74653, LC5, enx2
Reactivity
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(2), (5), (5), (16), (2), (2), (7), (4), (7), (4), (1), (4), (6), (2)
Application
Flow Cytometry (FACS), Fluorescence Microscopy (FM)
Pubmed 1 reference available
Quantity 100 tests
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Catalog No. ABIN411983
368.50 $
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Purpose Detects apoptosis in living cells by flow cytometry or fluorescence microscopy
Sample Type Cell Samples
Detection Method Fluorometric
Specificity The assay is based on the observation that soon after initiating apoptosis, most cell types translocate the membrane phospholipid phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface. Once on the cell surface, PS can easily be detected by staining with a fluorescent conjugate of Annexin V, a protein that has a strong natural affinity for PS.
Characteristics The one-step staining procedure takes only 10 minutes. Detection can be analyzed by flow cytometry or by fluorescence microscopy.
Components Annexin V-Cy5 (500 µL)
1X Binding Buffer (50 mL)
Alternative Name Annexin V
Research Area Cardiovascular, Atherosclerosis, Apoptosis/Necrosis, Coagulation
Application Notes Detection method: Flow cytometry (Ex/Em = 649 /670 nm) using Helium-Neon Laser and fluorescence microscopy
Protocol A. Incubation of cells with Annexin V-Cy5
1. Induce apoptosis by desired method.
2. Collect 1-5 x 10^5 cells by centrifugation.
3. Resuspend cells in 500 µL of 1X Binding Buffer.
4. Add 5 µL of Annexin V-Cy5. 5. Incubate at room temperature for 5 min in the dark. Proceed to B or C below depending on method of analysis.

B. Quantification by Flow Cytometry
Analyze Annexin V-Cy5 binding by flow cytometry (Ex = 649 nm, Em = 670 nm) using a Helium-Neon Laser.
For analyzing adherent cells, gently trypsinize and wash cells once with serum-containing media before incubation with Annexin V-Cy5 (A.3-5).

C. Detection by Fluorescence Microscopy
1. Place the cell suspension from Step A.5 on a glass slide. Cover the cells with a glass coverslip.
For analyzing adherent cells, grow cells directly on a coverslip. Following incubation (A.5), invert coverslip on glass slide and visualize cells. The cells can also be washed and fixed in 2 % formaldehyde before visualization. (Cells must be incubated with Annexin V-Cy3 before fixation since any cell membrane disruption can cause nonspecific binding of Annexin V to PS on the inner surface of the cell membrane.)
2. Observe the cells under a fluorescence microscope using Cy5 filter, or FITC/Cy3/Cy5 triple band filter (Chroma Technology) if performing triple labeling using these dyes, or detect cells using a CCD camera. Cells that have bound Annexin V-Cy5 will show bright red-blue staining on the plasma membrane.
Restrictions For Research Use only
Storage 4 °C
Expiry Date 12 months
Product cited in: Wang, Martin, Harris et al.: "Caspase inhibition blocks cell death and enhances mitophagy but fails to promote T-cell lymphoma." in: PLoS ONE, Vol. 6, Issue 5, pp. e19786, 2011 (PubMed).

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