Terry Riss, Rich Moravec, Nancy Murphy and Deborah Bishop
Promega Corporation, 2800 Woods Hollow Road, Madison WI 53711
We have developed a homogeneous fluorescent cytotoxicity assay that measures the release of lactate dehydrogenase (LDH) from cells with damaged membranes. The novel “add and read” homogeneous format of the CytoTox-ONE™ Assay (patent pending) was enabled by developing assay conditions that do not damage living cells, thus only LDH present in culture medium is detected and viable cells do not contribute to the signal. The CytoTox-ONE™ Assay procedure is to add reagent directly to cells, incubate for 10 min, stop the reaction, and record fluorescence. There is a linear relationship between the fluorescent signal and the number of damaged cells. The assay can detect fewer than 200 damaged cells in a 384-well format. The release of LDH from cells correlates inversely with viable cell number. The stability of the reagents and fluorescent signal are compatible with automation of the assay for HTS. Multiplexing of the CytoTox-ONE™ Assay with other assays will be described.