This article describes a method to assess NK cell-mediated cytotoxicity against cancer cells using calcein AM dye. The process involves incubating cancer cells with NK cells and measuring fluorescence to determine cytotoxic effects.
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.
To assess NK cell-mediated cytotoxicity using calcium AM, after growing human liver cancer cell line cells to a 70% to 80% confluency, as demonstrated, resuspend the cells in 3 milliliters of serum-free DMEM, and label the cells with 1.5 microliters of 10 millimolar calcium AM solution for 30 minutes at room temperature. At the end of the incubation, collect the cells by centrifugation, and wash the cells two times with 5 milliliters of PBS per wash.
Resuspend the cells at a 1 x 105 cells per milliliter of culture medium concentration, and add 100 microliters of cells to each well of a 96-well plate in triplicate per treatment condition. Next, add 1 x 105 human NK cells in 100 microliters of culture medium to each well of target cells, and place the plate in the cell culture incubator for four hours.
After the end of the incubation, capture at least 10 different fields of images of the calcium AM-positive cells for each replicate of each treatment condition on a fluorescence microscope at a 10 times magnification. Then, calculate the percent cytotoxicity using the formula.
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