Calcein Acetoxymethyl Staining to Evaluate Natural Killer 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.