Andrew Niles*, Tracy Worzella*, Richard Moravec*, William Daily**, Michael Scurria**, Laurent Bernad**, Brian McNamara*, Kay Rashka*, Deborah Lange*, and Terry Riss*.
*Promega Corporation, Madison, WI 53711 and **Promega Biosciences, San Luis Obispo, CA 93401
We have developed a homogeneous assay chemistry that simultaneously measures the relative number of live and dead cells in culture by detecting changes in cell membrane integrity. This single-addition assay measures two constitutive proteolytic activities; one is a marker of viability, and the other a marker of cytotoxicity. Together, these measures provide an inversely complimentary viability profile for each assay well. The resulting ratiometric assay data can also be used to improve additional multiplex endpoint data quality by response normalization, by increasing content, and by mitigating false negative or positive determinations. Here we detail advantages of the assay in simple cytotoxicity screening and in multiplexed cell-based models of necrosis and apoptosis. The functionality, sensitivity, and utility of the assay will be described using high density, multiwell formats with conventional fluid handling robotics and standard fluorometers or luminometers.