Abstract
Natural Killer (NK) cells are lymphocytes that are the first line of defense against malignantly transformed cells, virally infected cells and other stressed cell types. To study the cytolytic function of NK cells in vitro, a cytotoxicity assay is normally conducted against a target cancerous cell line. Current assay methods are typically performed in mixed 2D cocultures with destructive endpoints and low throughput, thereby limiting the scale, time-resolution, and relevance of the assay to in vivo conditions. Here, we evaluated a novel, non-invasive, quantitative image-based cytometry (qIBC) assay for detection of NK-mediated killing of target cells in 2D and 3D environments in vitro and compared its performance to two common flow cytometry- and fluorescence-based cytotoxicity assays. Similar to the other methods evaluated, the qIBC assay allowed for reproducible detection of target cell killing across a range of effector-to-target ratios with reduced variability. The qIBC assay also allowed for detection of NK cytolysis in 3D spheroids, which enabled scalable measurements of cell cytotoxicity in 3D models. Our findings suggest that quantitative image-based cytometry would be suitable for rapid, high-throughput screening of NK cytolysis in vitro, including in quasi-3D structures that model tissue environments in vivo.
•Quantitative image-based cytometry is a favored way to analyze NK cell cytolysis with minimized variance over other methods.•This method enabled non-invasive testing of cells at various effector to target cell ratios without a destructive endpoint.•NK cell cytolysis was found to be different in 2D and 3D environments which could be easily discerned by image-based analysis.•Modifying 3D spheroid formation to develop loose vs tight spheroids led to differences in effector cell killing.