Quantifying NK-cell Cytotoxicity Potentiated by Chimeric Antigen Receptors and Therapeutic Antibodies Using Live-cell...

Natural killer (NK) cells are innate lymphocytes that play a critical role in protective immunity against diverse intracellular pathogens and cancers. Their primary function is to kill target cells that are infected, malignantly transformed, or coated by antibodies via antibody-dependent cellular cytotoxicity (ADCC). NK cells can also be genetically engineered to express chimeric antigen receptors (CARs) that enable targeted recognition of specific antigens. Quantitative measurement of NK-cell cytotoxicity is essential for assessing baseline functionality and for preclinical evaluation of monoclonal antibodies and CAR-engineering strategies. However, in vitro functional assays remain highly variable across laboratories due to differences in cell preparation, target cells, effector-to-target ratios, co-incubation times, and readout methods, limiting reproducibility and cross-study comparisons. This article presents a standardized protocol for quantitative assessment of NK-cell cytotoxicity using flow cytometry and real-time, live-cell imaging. Primary human NK cells and CAR-expressing NK-92 cells were evaluated for their ability to kill cancer cells and antibody-coated target cells in a 96-well plate format to measure natural cytotoxicity, CAR-mediated killing, and ADCC. Target-cell survival was measured either continuously using live-cell imaging or at a defined time point by flow cytometry, which also enabled phenotypic characterization of NK cells and target cells. These protocols provide a robust framework using routine tissue culture, imaging, and flow cytometry methods to enable reproducible quantification of NK-cell effector functions for studies of innate immunity and NK cell-based immunotherapies.