This article describes a method for measuring the migration of natural killer (NK) cells in response to chemotactic stimuli. The protocol involves using a transwell permeable chamber and flow cytometry to quantify migrated cells.
Start by adding chemoattractants, such as a chemokine, in a multiwell plate.
Place a transwell permeable chamber of an appropriate pore size into the well. Add natural killer, or NK, cell suspension to the upper chamber and incubate.
A few chemokine molecules diffuse through the membrane and bind to NK cell receptors. This triggers signaling pathways that promote cytoskeletal changes, resulting in directional cell movement toward the higher chemoattractant concentration in the lower chamber.
After incubation, transfer a fixed volume of migrated cells from the lower chamber to a fluorescence-activated cell sorting or FACS tube. Add a predetermined number of fluorescent counting beads and perform FACS-based cell counting.
Using the following formula, determine the absolute number of migrated NK cells in the sample.
The dot plot gives the number of counting beads and cells separated as distinct regions, based on their fluorescence and scatter properties.
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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