To measure NK cell migration in response to chemotactic stimuli, collect human NK cells from a 70% to 80% confluent culture, and resuspend the cells at a 2.5 x 106 cells per milliliter of serum-free NK cell culture medium concentration. Next, add 600 microliters of serum-free medium containing the NK cell chemoattractant of interest per well.
And place one 6.5-millimeter diameter culture insert with 5-micrometer pores into each well of medium. Then, add 100 microliters of NK cells to the upper compartment of each insert, and place the plate in the cell culture incubator for four hours.
At the end of the incubation, transfer the entire volume of non-adherent migrated cells from the bottom of each well into individual 5-milliliter FACS tubes, and add 15 microliters of a predetermined number of flow cytometry counting beads to each tube. Then, using a flow cytometer, evaluate each cell sample according to standard flow cytometric analysis protocols, and calculate the absolute number of migrated NK cells using the formula.
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