Apoptosis Assays
Read the hallmarks of programmed cell death: executioner caspase activity, mitochondrial membrane potential and loss of membrane integrity.
Executioner caspases mark committed apoptosis. Fluorometric caspase-3/7 kits suit inducer and inhibitor screening.
JC-1 reports the collapse of mitochondrial membrane potential that occurs early in the intrinsic pathway.
Calcein-AM/PI double staining separates live from dead cells, and the ADP/ATP ratio tracks the shift from proliferation to cell death.
Apoptosis is a tightly regulated cell-death programme. Early on, phosphatidylserine flips to the cell surface (detected by Annexin V) and the mitochondrial membrane potential collapses (detected by JC-1). Executioner caspases 3 and 7 are then activated, and DNA is fragmented (detected by TUNEL).
Because these events occur in sequence, combining assays across the cascade gives the most reliable read-out of apoptosis versus necrosis.
Annexin V with a viability dye by flow cytometry and a caspase-3/7 activity assay are common starting points. Add TUNEL for DNA fragmentation or JC-1 for mitochondrial changes, depending on the stage of interest.
Pairing Annexin V with a membrane-impermeant viability dye separates early apoptotic cells (Annexin V positive, dye negative) from late apoptotic or necrotic cells (positive for both).
JC-1 is a dye that accumulates in healthy mitochondria. A loss of membrane potential shifts its fluorescence, giving an early indicator of apoptosis.