APOPTOSIS DETECTION

Annexin-V assay

During apoptosis, PS is translocated to the outer leaflet where it serves as a recognition molecule for macrophages. This occurs in the early phases of apoptotic cell death during which the cell membrane remains intact. Hapten (i.e. FITC or biotin) labelled annexin-V (green fluorescence) binds to PS residues exposed to the outer leaflet of the plasma membrane of apoptotic cells. Annexin-V does not bind to viable cells since the molecule is not able to penetrate the phospholipid layer. In necrotic cells the integrity of the membrane is lost and the inner leaflet of the membrane is available for binding of extrinsically applied annexin-V. To discriminate between necrotic and apoptotic cells, a membrane impermeable DNA stain such as propidium iodide (PI, red fluorescence) can be used simultaneously. This allows the discrimination of viable (FITC-PI-), early apoptotic (FITC+PI-) and necrotic cells or late apoptotic cells (FITC+PI+), which can be analyzed either by flow cytometry or fluorescence microscopy. 

Cell death Elisa assay