Quantitative high-content profiling of mitochondrial morphology with automated statistical analysis and integrated...

High-content screening (HCS) is a powerful approach for rapidly and efficiently assessing the harmfulness of numerous compounds across a wide range of cultured cell types. We recently developed a fully automated, miniaturized HCS wet-plus-dry pipeline (MITOMATICS) which leverages mitochondrial morphology as a sensitive and dynamic biomarker of cellular health or damage. Mitochondria are indeed not only vital for energy production and homeostasis, but also serve as critical gatekeepers of apoptotic cell death. MITOMATICS incorporates a proprietary software tool (MitoRadar) designed in-house to perform fast, comprehensive and cost-effective analysis of mitochondrial morphology in live cells. Together, the pipeline and its associated big data analytics software provide a valuable framework for early detection of acute mitotoxic effects of chemicals agents or physical stressors. To illustrate this, we present here a complete protocol for quantifying the impact of the pesticide chlorpyrifos-methyl on mitochondrial morphology of human lung epithelial BEAS-2B cells. Our results show that chlorpyrifos-methyl, even as a single compound, induces profound disruptions in mitochondrial subcellular structure. Beyond this case study, MitoRadar opens up promising avenues for investigating mitotoxicity across diverse cell types and environmental exposures, paving the way for a new generation of cellular diagnostics that could be of interest to the cell death community.