Calcein Acetoxymethyl Staining to Evaluate Natural Killer Cell-Mediated Cytotoxicity toward Cancer Cells

To begin, take a tube containing cancer cells in serum-free media to minimize serum esterase interference during the assay. Incubate with non-fluorescent calcein AM dye.

Active intracellular esterases hydrolyze calcein AM into membrane-impermeable fluorescent calcein dye retained in the cytoplasm.

Plate calcein-stained cancer cells and effector NK cells in the test wells, while the control wells only contain calcein-stained cancer cells without NK cells.

NK cells' activating cell surface receptors bind to activating ligands on the cancer cells, forming an immune synapse. This binding activates NK cell signaling, polarizing the cytotoxic granules containing perforins and granzymes toward the synapse for release.

Perforins form cancer cell membrane pores, facilitating the cellular entry of granzymes and initiating apoptosis. The compromised membrane integrity causes fluorescent calcein leakage, reducing cancer cell fluorescence.

Capture fluorescence images of the wells.

A decreased number of fluorescent live cancer cells in NK cell-containing wells than control wells indicates NK cell-mediated cytotoxicity toward cancer cells.