To prepare a cytotoxicity assay sample, first label 1.5-milliliter tubes for each sample and/or participant as appropriate, and pipette the desired ratio of NK cells, and DiO-labeled K562 cells into each tube.
Collect the cells by centrifugation, and carefully remove the supernatant without disturbing the cell pellet. Next, resuspend the cell mixture in 500 microliters of incomplete NK cell medium, and label the cells in each tube with 5 microliters of propidium iodide.
Collect the cells via another centrifugation, and incubate the cold culture at 37 degrees Celsius for two hours. At the end of the incubation, centrifuge the cells under the same centrifuge conditions, and resuspend the pellet in 25 microliters of fresh and complete NK cell culture medium.
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