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What is the Annexin V/PI assay?
Annexin V/PI assays are used to distinguish early apoptotic cells via Annexin V labeling and necrotic/dead cells via propidium iodide (PI) labeling. During the early stages of apoptosis, the cell membrane begins to lose its asymmetry, leading to the translocation of phosphatidylserine (PS) to the outer leaflet, where it can be detected by Annexin V conjugates. However, in cases of cell membrane rupture, such as in necrosis, Annexin V conjugates may penetrate and bind to PS residues on the inner leaflet, resulting in false-positive signals. Therefore, it is recommended to use a nuclear dye that does not permeate the cell membrane—such as PI—in combination with Annexin V conjugates. This allows users to differentiate between early apoptotic cells (stained with Annexin V), late apoptotic cells (stained with both Annexin V and PI), and necrotic cells (stained with PI alone).
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