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