title: Assessing Mitochondrial Bioenergetics in Isolated Mitochondria from Various Mouse Tissues Using Seahorse XF96 Analyzer.
abstract: Working with isolated mitochondria is the gold standard approach to investigate the function of the electron transport chain in tissues, free from the influence of other cellular factors. In this chapter, we outline a detailed protocol to measure the rate of oxygen consumption (OCR) with the high-throughput analyzer Seahorse XF96. More importantly, this protocol wants to provide practical tips for handling many different samples at once, and take a real advantage of using a high-throughput system. As a proof of concept, we have isolated mitochondria from brain, heart, liver, muscle, kidney, and lung of a wild-type mouse, and measured basal respiration (State II), ADP-stimulated respiration (State III), non-ADP-stimulated respiration (State IVo), and FCCP-stimulated respiration (State IIIu) using respiratory substrates specific to the respiratory chain complex I (RCCI) and complex II (RCCII). Mitochondrial purification and Seahorse runs were performed in less than eight working hours.
authors.affiliation: Institute of Human Genetics, Helmholtz Zentrum München, German Research Center for Enviromental Health, 85764, Neuherberg, Germany. arcangela.iuso@helmholtz-muenchen.de.|Institute of Human Genetics, Technische Universität München, Munich, Germany. arcangela.iuso@helmholtz-muenchen.de.
authors.forename: Arcangela
authors.initials: A
authors.lastname: Iuso
authors.affiliation: Institute of Human Genetics, Technische Universität München, Munich, Germany.
authors.forename: Birgit
authors.initials: B
authors.lastname: Repp
authors.affiliation: Institute of Human Genetics, Helmholtz Zentrum München, German Research Center for Enviromental Health, 85764, Neuherberg, Germany.|Institute of Human Genetics, Technische Universität München, Munich, Germany.
authors.forename: Caroline
authors.initials: C
authors.lastname: Biagosch
authors.affiliation: Institute of Human Genetics, Helmholtz Zentrum München, German Research Center for Enviromental Health, 85764, Neuherberg, Germany.
authors.forename: Caterina
authors.initials: C
authors.lastname: Terrile
authors.affiliation: Institute of Human Genetics, Helmholtz Zentrum München, German Research Center for Enviromental Health, 85764, Neuherberg, Germany.|Institute of Human Genetics, Technische Universität München, Munich, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Prokisch
mesh_terms: D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D004734:Energy Metabolism; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D009928:Organ Specificity; D010101:Oxygen Consumption; D013223:Statistics as Topic / Q000379:methods
keywords: High-throughput; Mitochondrial coupling assay; Mouse tissues; Respiratory control ratio (RCR); Seahorse XF96