A real‐time digital bio‐imaging system to quantify cellular cytotoxicity as an alternative to the standard chromium‐51 release assay

Summary Reliable measurement of cellular cytotoxicity is essential for the characterization of immune responses and for the monitoring of antibody treatment efficacy. Until now, the standard 51Cr‐release assay has remained the sole sensitive assay that measures cellular cytotoxicity. Alternative non‐radioactive assays have been developed but they do not provide accurate measurement of target cell cytotoxicity. The cost and hazard of handling radioactivity are strong incentives to find alternative solutions to 51Cr. We took advantage of the recent development of cell‐imaging multimode readers to develop a novel non‐radioactive and real‐time cytotoxic assay that demonstrates good reproducibility and sensitivity. The extent of target‐cell cytotoxicity is monitored over time by imaging and quantifying live fluorescent target cells in 96‐well plates. We have developed classical natural killer cell assays in the presence or absence of blocking antibodies and antibody‐dependent cell‐mediated cytotoxicity. We show that in these assays, cell killing occurs within the first 2 hr with half maximum killing reached after 30 min. This technology has numerous applications such as natural killer and T‐cell cytotoxicity assays and can be extended to cell survival and apoptosis measurement assays. We have developed a novel non‐radioactive cell‐mediated cytotoxic assay that provides a real‐time measurement of cellular cytotoxicity based on accurate quantification of fluorescent target cells using the cell imaging multi‐mode plate reader ‘Cytation™ 5’. We established that this assay is suitable for standard natural killer cell assays such as measurement of natural cytotoxicity and antibody‐dependent cell‐mediated cytotoxicity as well as cytotoxic T lymphocyte assays. This assay provides an effective alternative to the standard 51Cr‐release assay.