Abstract
Abstract Recently developed assay technologies have made it possible to use a multi-well plate reader to estimate the number of live or dead cells in culture in real time over a period of days to monitor the effects of long term exposure to test substances. An advantage of the real time assay chemistries is that the cells remain viable and available for other measurements. The purpose of this study was to determine if the real time assay methods could be multiplexed with other assay endpoints and applied to three dimensional (3D) culture models. The measurement of the number of live cells in real time is accomplished by providing a pro-substrate that viable cells (but not dead cells) can convert into a substrate for a luciferase derived from a marine shrimp. The pro-substrate and luciferase can be added with a single reagent addition step and because they are not toxic, they can be incubated with cells in culture for up to three days. Live cell conversion of the pro-substrate into a substrate for luciferase results in a luminescent signal that is proportional to the number of live cells. The signal diminishes immediately upon cell death. Detection of dead cells in real time is accomplished by using a fluorogenic DNA binding dye that is non-permeant and thus non-toxic to live cells. The dye enters dead cells that have a compromised membrane, binds to DNA, and becomes fluorescent. We have demonstrated that the real time assays can be multiplexed to measure both live and dead cells in the same sample. We further demonstrate the use of these assays to measure viability of cells in 3D spheroids formed using the hanging drop culture technique. We also demonstrate multiplexing of real time assays with subsequent measurement of apoptosis or extraction of RNA for expression studies. Utilization of real time assay chemistries that are non-toxic enabled repeated recording of data in a kinetic mode from the same sample of cells which eliminated the need for duplicate cultures and provided flexibility during assay protocol development. The ability to multiplex real time assays improved overall lab efficiency by eliminating the need for duplicate cultures for each experiment. Citation Format: Terry Riss, Jolanta Vidugiriene, Sarah Duellman, Wenhui Zhou, Gediminas Vidugiris, Mike Valley, Jean Osterman, Ruslan Arbit, Laurent Bernad, Poncho Meisenheimer, James J. Cali. Continuous real-time measurement of live and dead cells in culture over multiple days. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5446. doi:10.1158/1538-7445.AM2015-5446
Published Version
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