The Xpert Annexin V-FITC Apoptosis Detection Assay offers a simple, fast and reliable method for the fluorescent detection of apoptotic cells and quantitative determination by flow cytometry. This assay is based on binding of FITC-labeled Annexin V to phosphatidylserine sites (PS) present on the membrane surface of apoptotic cells combined with the binding of propidium iodide (PI) to cellular DNA in necrotic cells in which cell membrane integrity has been completely compromised. This allows to distinguish between viable cells, early apoptotic cells and necrotic cells.
Apoptosis is a gradual process leading to cell death, characterized by specific morphologic features. The translocation of PS from the inner to the outer leaflet of the plasma membrane, hence exposing PS to the external cellular environment, is one of the earliest phenomena of apoptosis, preceding loss of membrane integrity that occurs in later stages of apoptosis or during necrosis. Therefore when using a combination of green fluorescent FITC-labeled Annexin V (Annexin A5) and red fluorescent PI, viable cells are negative for both, whereas early apoptotic cells are Annexin V-positive, as Annexin V binds strongly to PS, even when labeled with FITC, but PI-negative (as intact membranes are impermeable to PI). Cells entering later stages of apoptosis or necrosis will be both Annexin V and PI-positive.
Albeit that this assay does not distinguish between cells that have died as a result of apoptosis and cells that have suffered another necrotic pathway, by measuring apoptosis over time, cells can often be followed from Annexin V-negative/PI-negative via Annexin V-positive/PI-negative to Annexin V-positive/PI-positive. Encountering all these three phenotypes at the same time within a cell population, or observing a shift through these three stages within a cell population over time, is thus a strong indicator of apoptosis.