Abstract 5532: A pro-fluorescent membrane integrity dye for the real-time assessment of cytotoxicity.

Abstract

Abstract Cell-based assay models are now universally employed as a routine means of establishing and ranking on- and off-target toxicities. It is well appreciated, however, that individual cytotoxic phenotypes are shaped not only by compound dosage, but by the length of compound-cell exposure. In addition to compound-specific attributes, inherent cellular factors such as biotransformation capacity, cell cycle susceptibility, and receptor expression often dictate the kinetics of cytotoxicity. Unfortunately, the majority of conventional cytotoxicity assays are performed at a terminal endpoint (48-72h) which at worst can underestimate cytotoxicity due to biomarker degradation, or at best, fail to reveal important features of the cytotoxic event which may be important for establishing mechanism-of-action. We have developed a pro-fluorescent, cell-impermeant probe which can be applied to cells at the time of dosing to report changes in membrane integrity as they occur in real-time. The dye enters cells with impaired membrane integrity, greatly enhancing fluorescence which is proportional to non-viable cell number. The dye can be used in traditional plate based formats using standard fluorometry or with image based readers (Essen Bioscience Incucyte™) to establish the dose-dependent kinetics of cytotoxicity. Here we describe the dye's utility using mechanistically distinct cytotoxins with both cancer cell lines (on-target) and terminally differentiated cells (off-target) and correlate this dead cell fluorescence activity to other cytotoxicity biomarkers by same-well multiplexed methods. Citation Format: Richard L. Somberg, Andrew Niles, Tracy Worzella, Dan Lazar. A pro-fluorescent membrane integrity dye for the real-time assessment of cytotoxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5532. doi:10.1158/1538-7445.AM2013-5532