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 these challenges and successfully measured MMP changes across multiple cell lines and four different human primary cells. This no-wash assay has been miniaturized into a 384-well format. It is robust, with a Z' value > 0.5 on both HepG2 and HeLa cells, making it suitable for high-throughput screening. The reagents in the m-MPI homogeneous assay kit are non-toxic, as was demonstrated in HepG2 and HeLa cell lines using Dexorgen's EnerCount ATP assay. Using this assay, we conducted a pilot screen on human hepatocytes against the Library of Pharmacologically Active Compounds (LOPAC) (Sigma). Identified hits were subsequently confirmed through follow-up studies. Collectively, these findings establish the m-MPI assay as a reliable, scalable tool for mitochondrial functional analysis and drug discovery.