The aim of this assay is to assess the effects of the test item on NK cells.
NK cells were named as „natural killers” as they do not require activation to kill cells that are missing "self" markers of MHC1. In addition, CD16 expression on NK cells plays a critical role in the antibody-dependent cell-mediated cytotoxicity by the recognition of cell surface-bounded IgG.
In vitro study using human PBMCs or purified NK cells as effector cells and K562 cell line as target cells.
Principle of the study: K562 is a human erythroleukemia cell line which is widely used to measure NK cell cytotoxic activity as they lack class I MHC expression required to inhibit NK activity. The assay will be carried out by co-culturing of whole blood, human PBMCs or purified NK cells as effector cells in the presence or absence of test item with fluorescently-labeled K562 cell line target cells which are sensitive to NK cell-mediated cytotoxicity. The number of K562 target cells killed by NK cells can be determined by flow cytometry using a fluorescent dead cell dye.
To induce NK cell-mediated cytotoxicity, CFSE-stained K562 cells are co-cultured with effector cells (whole blood/PBMCs/purified NK cells) for various time points using different effector:target cell (E:T) ratios. Both effector and target cells are treated separately with the test item to distinguish background cell death from the test item originated side effects.
Dead cells are stained after the incubation period with the dead cell dye 7-AAD and samples are subjected to flow cytometry analysis. CFSE+7-AAD+ double positive cell population represents the dead cells indicating the cytotoxic effect of NK cells.