Abstract 4062: A kinetic live-cell imaging approach to measuring cytotoxicity in vitro

Cytotoxicity assays play a critical role in the identification of potential anti-cancer chemotherapeutic agents. Commonly used in vitro cytotoxicity assays evaluate a range of end point parameters such as the release of lactate dehydrogenase (LDH) and glutathione (GSH) following membrane rupture, generation of reactive oxygen species (ROS), cell proliferation, and disruption of mitochondrial trans-membrane potential. Critical factors contributing to the predictive nature of these assays include compound concentration, and more importantly, the time allowed for the compound to elicit an effect. Although these multiplexed assays are able to simultaneously measure multiple indicators of in vitro cytotoxicity, they typically assess a single time point and are unable to assess the biological activity over time. We present data showing that we are able to kinetically measure cell wall integrity, while simultaneously monitoring cell death associated morphological changes using a commercially available cell impermeable DNA dye, YOYO-1, coupled with the IncuCyte™ FLR live-cell imaging system. This no-wash, mix-and-read assay provides a statistically robust method to study the kinetic induction of cytotoxicity in vitro. Specifically we use HT 1080 fibrosarcoma cells, MDA-MB-231 breast adenocarcinoma cells, and HeLa cervical adenocarcinoma cells in conjunction with the cytotoxic agents staurosporine and camptothecin, and the cytostatic agent, cycloheximide. These data illustrate the ability to kinetically detect, quantify, and analyze cytotoxicity data in a 96-well plate format using images collected in real time. We show that the quantitative data obtained in this study is amenable to moderate throughput screening protocols with Z’ values >0.6 achieved in both intra- and inter-assay reproducibility studies. We also provide evidence that that the high definition phase contrast images provide qualitative verification of quantitative results allowing for the discrimination between cytotoxic and cytostatic compounds. This strategy combines automated data acquisition and analysis to provide an informative and efficient approach to detecting pharmacological or genetic manipulations that alter cell viability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4062. doi:1538-7445.A