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P1014
The Use of Multi-Parametric High-Content Screening Assays to Monitor Cytotoxicity & Different Programmed Cell Death Pathways.
Presenter Krishna Vattem, Thermo Fisher Scientific
Additional Authors: Bhaskar S. Mandavilli, Richik N. Ghosh
Apoptosis, one form of programmed cell death, is characterized by morphological changes such as loss of mitochondrial membrane potential, induction of nuclear condensation and cell permeability. Mitochondrial-dependent apoptosis is accomplished by release of cytochrome c from mitochondria, cleavage of caspase 9, caspase 3 and PARP, and finally nuclear condensation and nuclear fragmentation. While the mechanism of apoptosis is mainly caspase-dependent, there are alternative forms of programmed cell death that can be induced in a caspase-independent fashion, such as in autophagy. Autophagy is a catabolic process involving sequestration and self-ingestion of various cellular constituents such as organelles and long-lived proteins. Cells that undergo excessive autophagy are triggered to die in a non-apoptotic manner. Automated, quantitative, cell-based imaging high-content screening (HCS) assays enable simultaneous measurements of multiple targets in individual cells, allowing different programmed cell death pathways such as apoptosis and autophagy to be distinguished and characterized. We performed a panel of cell-based HCS assays using a Thermo Scientific Cellomics® ArrayScan® HCS Reader and various Thermo Scientific Cellomics HCS Reagent Kits. These tools enabled us to characterize the cytotoxicity triggered by different compounds by simultaneously measuring the intracellular changes in nuclear morphology and various apoptotic and autophagic markers. Time- and dose-dependent responses were assayed, and compounds were ranked according to their ability to induce cytotoxicity. These multi-parametric assays for simultaneously measuring changes in mitochondrial membrane potential, nuclear morphology, cell permeability, induction of apoptosis and autophagy validate HCS as a useful and efficient platform to profile cell health, as well as to characterize the possible alternative mechanisms of cell death.