An In Vitro Assay for Evaluating Natural Killer Cell Cytotoxicity against Cancer Cells

Take a multi-well plate containing natural killer, or NK, cells and cancer cells. Control wells contain only cancer cells.

Centrifuge to facilitate cellular contact.

Incubate. Activating receptors on NK cells bind to cancer cell surface ligands, triggering NK cells to release granules containing cytotoxic molecules.

These molecules create cancer cell membrane pores and induce apoptosis releasing intracellular contents, including lactate dehydrogenase, or LDH.

Add a detergent to lyse the cancer cells in control wells for maximum LDH release.

Centrifuge and transfer a portion of LDH-containing supernatants to an assay plate.

Add the assay reagent and incubate.

LDH converts lactate to pyruvate reducing NAD+. Diaphorase uses NADH  to reduce tetrazolium salt, forming a red-colored formazan product.

Add an acidic solution to stop the enzymatic reaction.

Use a microplate reader to measure the formazan absorbance in the wells.

The formazan intensity is proportional to the number of lysed cancer cells in the test well,  indicating NK cell cytotoxicity against cancer cells.