Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS) from all mammalian species. Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by fluorescently labeled Annexin V in a calcium-dependent manner. In early-stage apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI), 7-AAD, or Fixable Viability Dyes such as eFluor® 660 or eFluor® 780. These cells will stain with Annexin V but not a viability dye, thus distinguishing cells in early apoptosis. However, in late stage apoptosis, the cell membrane loses integrity thereby allowing Annexin V to also access PS in the interior of the cell. A viability dye can be used to resolve these late-stage apoptotic and necrotic cells (Annexin V, viability dye-positive) from the early-stage apoptotic cells (Annexin V positive, viability dye-negative). Not included: Propidium Iodide Staining Solution (cat. 00-6990) 7-AAD Viability Staining Solution (cat. 00-6993) Note: Fixable Viability Dye eFluor® 450 is not recommended for use with Annexin V Apoptosis Detection Kits.
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