An In Vitro Assay to Study the Cytotoxic Capability of Cytokine-Induced Killer Cells

Take a culture of cancer cells with fluorescently labeled cytoplasmic proteins.

Add cytokine-induced killer or CIK cells — effector T cells with natural killer cell-like characteristics — possessing anti-tumor activity.

Natural killer group 2 member D or NKG2D receptors on the CIK cells bind to stress-inducible proteins on the cancer cells.

The binding induces CIK cells to release cytoplasmic granule toxins — perforins and granzymes.

Perforins form pores in the cancer cell membrane, allowing the entry of granzymes, which activate caspases, inducing apoptosis.

Post-incubation, harvest the cells. Add a fluorescent nucleic acid-binding cell viability dye.

The dye enters through the damaged cell membrane and binds to DNA, resulting in dual-stained dead cells.

With an intact cell membrane, live cells are impermeable to the dye.

Pass the cells through a flow cytometer to detect live cells displaying single fluorescence and dead cells with dual fluorescence.

Create a scatter plot to measure the percentage of dead cancer cells, quantifying the cytotoxic effect of CIK cells.

To perform the cytotoxic assay, co-culture CIK and human chronic myeloid leukemia K562 cells, by adding 1 milliliter of K562 cells to each well in a 6-well plate at a density of 0.5 million cells per milliliter. Then, add 1 milliliter of basal medium with or without CIK cells to the 6-well plate, as listed in the text protocol. Mix the cell suspensions by gently pipetting up and down at least three times.

Place the plate in the incubator for 24 hours. Now, co-culture CIK and ovarian OC-3 cells by adding 1 milliliter of basal medium with or without CIK cells to the 6-well plate, as listed in the text protocol. Mix the cell suspensions by gently pipetting up and down at least three times with the blade in the incubator for 24 hours. Following incubation, harvest the CIK-K562 cell suspension directly into a 15-milliliter sterile tube.

To harvest both the suspension and adherent cells from the CIK-OC-3 groups, first, transfer the cell suspension to a 15-milliliter sterile tube. Wash the well with 1 milliliter of sterile PBS. Collect the PBS, and add it to the tube. Then, add 0.5 milliliters of cell dissociation enzyme solution, and incubate for five minutes at 37 degrees Celsius.

Add 1 milliliter of the solution from the same tube to the corresponding well, and gently mix the cells by pipetting up and down at least three times with a 1-milliliter sterile pipette. Collect all the cells in the same tube. Centrifuge at 300 x g for 10 minutes. Aspirate the supernatant, and resuspend the cells in 1 milliliter of sterile PBS. Pellet the cells at 300 x g for 10 minutes. Then, aspirate the supernatant, and resuspend the cells in 100 microliters of sterile PBS.

Add 5 microliters of 7-AAD dye to the cell suspension. Gently mix the cells by pipetting up and down at least three times with a 1-milliliter sterile pipette. Incubate for 10 minutes, and leave in the dark before analysis.

To perform the cytolytic capability assay, mix the cell suspension and repeat the preparation for flow cytometry. Click the New Specimen button to add a specimen and tube to the experiment. Name the tubes as listed in the text protocol.

To create a scatter gating system for the cytolytic assay, first, select Tube 1 and click on the Dot Plot button. To create an FSC-A/SSC-A plot, draw a rectangle gate over all events with an FSC-A threshold greater than 50,000 to exclude cell debris. Select the SSC-A/CFSE parameter for the new dot plot. Then, select the 7-AAD/CFSE parameter for the new dot plot, and draw a four-quadrant gate to define the four subpopulations.

Click the Load Sample button to analyze the blank control sample first. Adjust the voltage of SSC-A and FSC-A. Identify the dead cell population by using the CFSE and 7-AAD channel parameters. Record the data from greater than 20,000 CFSE-positive cells in each specimen. Open the files containing the statistical values of each individual specimen to analyze the non-viable cell populations, and export the data into analysis files.