An In Vitro Assay for Evaluating Natural Killer Cell Cytotoxicity against Cancer Cells

First, obtain a round-bottom culture-treated 96-well plate, and set it up as outlined in table 1 of the text protocol. Centrifuge the assay plate at 250 times g for 4 minutes to be certain that the effector and target cells are in contact. Next, incubate the plate in a humidified chamber at 37 degrees Celsius with 5% carbon dioxide for 5 hours.

45 minutes prior to harvesting the supernatants, add 10 microliters of 10x lysis solution to the target cell maximum LDH release wells, and return the plate to the humidified chamber. When the incubation is complete, centrifuge the plate at 250 times g for 4 minutes. Then, use a multichannel pipettor to transfer 50-microliter aliquots from every well to a fresh 96-well flat-bottom assay plate.

Add 50 microliters of assay reagent to each well of the assay plate. Cover the plate with foil to protect it from light, and incubate at room temperature for 30 minutes. After this, add 50 microliters of stop solution to each well. Make sure to read the plate within 1 hour of adding the stop solution and record the absorbance at 490 nanometers.