Characterization of the Mitochondria Function and Metabolism in Skin Fibroblasts Using the Biolog MitoPlate S-1

S-1 MitoPlates™ from Biolog enable the characterization of mitochondria's function in live cells by measuring the rates of electron flow into and through the electron transport chain from different NADH or FADH₂ producing metabolic substrates. This technology uses 96-well microplates pre-coated with triplicate repeats of a set of 31 substrates. Those 31 metabolic substrates have different routes of entry into the mitochondria, use different transporters, and are also oxidated by different dehydrogenases, producing reducing equivalents in the form of NADH or FADH₂. The electrons produced upon oxidation of NADH or FADH₂ at complex I or II, respectively, then travel to cytochrome c, where a tetrazolium redox dye (MC) can act as terminal acceptor, turning purple and absorbing at 590 nm. This mechanism allows the evaluation of cellular substrate preference by following the kinetics of MC reduction in the presence of selected substrates.In this chapter, we describe the step-by-step protocol to prepare an experiment using MitoPlate S-1 array and the OmniLog instrument to assess the metabolism of human dermal fibroblasts. We also give detailed information on how to analyze the raw data generated by the Biolog Data Analysis software to extract meaningful information and produce useful data visualizations, using reproducible methods based on a single structured dataset.