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Annexin V-APC Apoptosis Detection Kit

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Detection Method Fluorometric
Background Annexin A5 (or Annexin V) is a cellular protein in the annexin group. Annexin A5 is used as a probe to detect cells that have expressed phosphatidylserine (PS) on the cell surface, an event found in apoptosis as well as other forms of cell death. The annexin A5 affinity assay typically uses a conjugate of annexin V and a fluorescent or enzymatic label, biotin or other tags, or a radioelement, in a suitable buffer (annexin V binding to PS is Ca2+ dependent). The assay combines annexin V staining of PS membrane events with the staining of cell nucleus with PI or AAD-7 in living cells to distinguish from dead cells. Annexin V Apoptosis Detection Kit is based on the observation that soon after initiating apoptosis, cells translocate the membrane phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface. Once on the cell surface, PS can be easily detected by staining with a fluorescent conjugate of Annexin V, a protein that has a high affinity for PS. Detection can be analyzed by flow cytometry or by fluorescence microscopy.
Protocol 1. Dilute the Binding buffer (10×) to work solution (1×) with distilled water. 2. Harvest cell, then wash once with cold PBS, remove the PBS from the cell pellet. 3. Wash again with cold 1× Binding buffer, remove the Binding buffer from the cell pellet. Resuspend cells in cold 1× Binding buffer to a concentration of 1×106 cells/mL. 4. Add 100myL of cells (1×10^5) to each appropriate tube. 5. 6. Add 5myL of Annexin V-APC to appropriate tubes. 7. Gently vortex each tube and incubate for 10 minutes at room temperature in dark. 8. Add 5myL of PI solution and incubate for 5 min at room temperature in dark. 9. Wash cells once in PBS and resuspend in PBS. 10. Analyze by flow cytometry within 4 hours.
Restrictions For Research Use only
Storage 4 °C
Product cited in: Koopman, Reutelingsperger, Kuijten et al.: "Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis." in: Blood, Vol. 84, Issue 5, pp. 1415-20, 1994 (PubMed).

Vermes, Haanen, Steffens-Nakken et al.: "A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V." in: Journal of immunological methods, Vol. 184, Issue 1, pp. 39-51, 1995 (PubMed).