Abstract 3743: Bioluminescent, non-lytic, real-time cell viability assay

Abstract

Abstract Cell-based assay development for cancer drug discovery continues to advance toward more innovative methods that provide efficient workflow and meaningful data. Rapid, sensitive techniques that are non-lytic and therefore facilitate multiplexing with a wide range of assays allow more data to be gained per well and conserve precious samples. Real time measurement of cell viability also allows numerous measurements over time without the need for separate plates at each time point. Real time measurements allow unique analyses including interrogation of the timing of drug killing, identification of static vs toxic drug effects, and determination of the ideal time to multiplex an additional assay. Cancer drug discovery would benefit from novel assay technologies that allow real time measurements with multiplexing capability. We have developed a homogeneous, non-lytic, and bioluminescent method to analyze cell viability in real time through measurement of the reducing potential of the cell. The signal correlates with the number of viable cells making it well-suited for cytotoxicity studies. This Real Time Cell Viability Assay utilizes the NanoLuc luciferase enzyme, which is 100-fold brighter than either firefly (Photinus pyralis) or Renilla reniformis luciferase, and the novel substrate, furimazine. Together these reagents produce high intensity luminescence through an ATP-independent reaction. The assay can be performed in two formats, live cell endpoint or continuous read. The live cell endpoint method is sensitive, rapid, and enables extensive multiplexing opportunities due to the non-lytic measurement and signal depletion upon cell lysis. When the assay is set up in the continuous read format, the cell viability reagents can be added at the same time as cell plating, drug dosing, or at whatever point in the assay the researcher would like to start obtaining viability readings. The luminescent signal can be continually monitored from the same wells over an extended period of time to analyze cell viability in real time. This real time cell viability assay enables non-lytic cell viability measurements, extensive multiplexing options including both fluorescent and luminescent assays with no special filter requirements, straightforward normalization studies, conservation of precious samples and reagents, and a sensitive, real time measurement of cell viability. Citation Format: Sarah J. Duellman, Jolanta Vidugiriene, Wenhui Zhou, Jean Osterman, Ruslan Arbit, Laurent Bernad, Poncho Meisenheimer, James J. Cali. Bioluminescent, non-lytic, real-time cell viability assay. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3743. doi:10.1158/1538-7445.AM2014-3743