As a new method for detecting apoptosis, the Annexin Vbinding assay is based on the measurement of the loss of plasma membraneasymmetry. Under normal physiological conditions, cell sustain a strictly asymmetricdistribution of phospholipids in the two leaflets of the cellular membranes,where phosphatidylserine (PS) facing the cytosolic side. In the early stages ofapoptosis, this asymmetry disappeared rapidly, while the integrity of themembrane remained, which resulted in the exposure of PS to the outer leaflet bymeans of phagocytosis. This phenomenon can be detected by hapten-labeled AnnexinV, which shows high affinity for PS residues in the presence of micromolarconcentrations of Ca²⁺.By simultaneously excluding nuclear dye propidium iodide (PI), apoptotic cellsand necrotic cells are distinguished.

Incubation of Cellswith Annexin V-FITC and PI

Ÿ   Inducingcell apoptosis according to your experimental protocol.

Ÿ   Suspensioncells are collected with the culture supernatant, pelleted by centrifugationand washed twice, while adherent cells are harvested by mechanical scraping orgently trypsinizing and washed with serum-containing media.

Ÿ   Suspensionand adherent cells are finally pooled and resuspended in culture medium to afinal concentration of 1×10⁶ cells/mL.

Ÿ   Add annexinV-FITC and PI to a final concentration of 3 μg/mL and 5 μg/mL, the cells aresubsequently incubated for 10 min in the dark.

Flow Cytometry

At least 10,000 cells per sample are analyzed. Thewavelength of excitation light is 488 nm, and the emission filters are 515-545nm (green, FITC) and 600 nm (red, PI). Using electronic compensation toeliminate the bleed through of fluorescence.