The markers commonly used to detect apoptotic cells include:
Phosphatidylserine Translocation using Annexin V Assay
During early apoptosis, phosphatidylserine (PS) translocates or externalizes from the inner to the outer membrane leaflet, preceding other apoptotic events such as DNA fragmentation and loss of membrane integrity. Annexin V has a high affinity for phosphatidylserine, which makes it very useful for detecting apoptotic cells. The externalization of PS can be easily detected using fluorescently labeled Annexin V. While Annexin V can also bind to PS on late apoptotic and necrotic cells, co-staining with a membrane-impermeable dye such as propidium iodide (PI) helps distinguish early apoptotic cells from late apoptotic cells with compromised membrane integrity.
Mitochondrial Potential Assay using JC-1 Dye
Apoptosis is preceded by mitochondrial membrane potential loss. The lipophilic cationic dye JC-1 accumulates in the mitochondrial matrix of healthy cells due to their intact membrane potential, emitting red fluorescence. However, in apoptotic cells, the loss of membrane potential results in monomeric JC-1, emitting green fluorescence. This property enables the differentiation of healthy (red) and apoptotic (green) cells, providing a straightforward fluorescent-based method.
Caspase Activity Assay – FLICA Assay
The Fluorochrome-Labeled Inhibitor of Caspases Assay (FLICA) provides a reliable means to detect apoptotic cells based on caspase activity. Caspases, a family of protease enzymes essential for carrying out programmed cell death. Upon activation, caspases mediate apoptosis by proteolysis of specific substrates. FLICA measures the activity of caspase 3/7, 8, or 9 using a caspase-specific inhibitor sequence linked to a fluorescent probe. The fluorescence intensity directly correlates with the amount of active caspase present in viable cells. Co-staining with propidium iodide allows for the identification of non-viable cells.