Cytotoxic and chemotactic dynamics of NK cells quantified by live-cell imaging.

Natural Killer (NK) cells detect and eliminate virus-infected cells and cancer cells, and are crucial players of the human immune defense system. Although the relevant molecular machineries involved in NK cell activation and NK-target cell interactions are largely known, how their collective signaling modulates the dynamic behaviors of NK cells, e.g., motility and cytotoxicity, and the rate-limiting kinetics involved are still in need of comprehensive investigations. In traditional bulk killing assays, heterogeneity and kinetic details of individual NK-target cell interactions are masked, seriously limiting analysis of the underlying dynamic mechanisms. Here we present detailed protocols of a number of live-cell imaging assays using fluorescent protein reporters and/or a live-cell dye that enable the acquisition of quantitative kinetic data at the single cell level for elucidating the mechanism underlying the interaction dynamics of primary human NK cells and epithelial cancer cells. Moreover, we discuss how the imaging data can be analyzed either alone or in combination to quantify and determine the key dynamic steps/intermediates involved in specific NK cell activity, e.g., NK cell cytotoxic modes and their associated kinetics, and NK cell motility toward different cancer targets. These live-cell imaging assays can be easily adapted to analyze the rate-limiting kinetics and heterogeneity of other cell-cell interaction dynamics, e.g., in T cell function.