A Novel No-Wash (Homogenous) High-Throughput Toxicity Assay on Primary Cells

Mitochondrial membrane potential (MMP) is a critical marker of mitochondrial and, therefore, cellular function. However, current dyes used for MMP measurement possess significant limitations: nonspecific targeting, long incubation times, potential mitochondrial toxicity, poor water solubility, and numerous wash steps. Using a novel fluorescent dye—mitochondrial membrane potential indicator (m-MPI)—in combination with a masking dye, we overcame said limitations and successfully measured MMP changes on different cell lines, as well as four different human primary cells. This no-wash assay has been miniaturized into a 384-well format. It is robust with Z’ value > 0.5 on both HepG2 and HeLa cells and can be used for high throughput screening. The reagents in the m-MPI homogeneous assay kit are not toxic to cells, as was demonstrated on HepG2 and HeLa cell lines using Dexorgen’s EnerCount ATP assay. Using this assay, we conducted a mini screening on human hepatocytes against the Library of Pharmacologically Active Compounds (LOPAC) (Sigma). The resulting hits were then confirmed with follow-up studies.