title: Efficient isolation of pure and functional mitochondria from mouse tissues using automated tissue disruption and enrichment with anti-TOM22 magnetic beads.
abstract: To better understand molecular mechanisms regulating changes in metabolism, as observed e.g. in diabetes or neuronal disorders, the function of mitochondria needs to be precisely determined. The usual isolation methods such as differential centrifugation result in isolates of highly variable quality and quantity. To fulfill the need of a reproducible isolation method from solid tissues, which is suitable to handle parallel samples simultaneously, we developed a protocol based on anti-TOM22 (translocase of outer mitochondrial membrane 22 homolog) antibody-coupled magnetic beads. To measure oxygen consumption rate in isolated mitochondria from various mouse tissues, a traditional Clark electrode and the high-throughput XF Extracellular Flux Analyzer were used. Furthermore, Western blots, transmission electron microscopic and proteomic studies were performed to analyze the purity and integrity of the mitochondrial preparations. Mitochondrial fractions isolated from liver, brain and skeletal muscle by anti-TOM22 magnetic beads showed oxygen consumption capacities comparable to previously reported values and little contamination with other organelles. The purity and quality of isolated mitochondria using anti-TOM22 magnetic beads was compared to traditional differential centrifugation protocol in liver and the results indicated an obvious advantage of the magnetic beads method compared to the traditional differential centrifugation technique.
authors.affiliation: Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany.
authors.forename: Andras
authors.initials: A
authors.lastname: Franko
authors.affiliation: Institute of Vegetative Physiology, Medical Faculty, University of Köln, Köln, Germany.
authors.forename: Olivier R
authors.initials: OR
authors.lastname: Baris
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
authors.forename: Eva
authors.initials: E
authors.lastname: Bergschneider
authors.affiliation: Research Unit Protein Science, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: von Toerne
authors.affiliation: Research Unit Protein Science, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany.
authors.forename: Stefanie M
authors.initials: SM
authors.lastname: Hauck
authors.affiliation: Research Unit Analytical Pathology, Institute of Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany.
authors.forename: Michaela
authors.initials: M
authors.lastname: Aichler
authors.affiliation: Research Unit Analytical Pathology, Institute of Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany.
authors.forename: Axel K
authors.initials: AK
authors.lastname: Walch
authors.affiliation: Institute of Developmental Genetics, Helmholtz Zentrum München, Technische Universität München, Neuherberg, Germany ; Max Planck Institute of Psychiatry, Munich, Germany ; Technische Universität München, Lehrstuhl für Entwicklungsgenetik, c/o Helmholtz Zentrum München, Neuherberg, Germany ; DZNE - Deutsches Zentrum fuer Neurodegenerative Erkrankungen, Site Munich, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wurst
authors.affiliation: Institute of Vegetative Physiology, Medical Faculty, University of Köln, Köln, Germany ; Center for Molecular Medicine (CMMC), University of Köln, Köln, Germany ; Cologne Excellence Cluster on Cellular Stress Responses in Ageing-associated Diseases (CECAD), University of Köln, Köln, Germany.
authors.forename: Rudolf J
authors.initials: RJ
authors.lastname: Wiesner
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
authors.forename: Ian C D
authors.initials: IC
authors.lastname: Johnston
authors.affiliation: Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany ; Technische Universität München, WZW - Center of Life and Food Science Weihenstephan, Chair of Experimental Genetics, Freising-Weihenstephan, Germany ; German Center for Diabetes Research (DZD), Neuherberg, Germany.
authors.forename: Martin Hrabĕ
authors.initials: MH
authors.lastname: de Angelis
mesh_terms: D000818:Animals; D001331:Automation; D002458:Cell Fractionation / Q000379:methods*; D002498:Centrifugation; D060328:Magnetic Phenomena; D051379:Mice; D008863:Microspheres; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D008930:Mitochondria, Liver / Q000378:metabolism / Q000648:ultrastructure; D008931:Mitochondria, Muscle / Q000378:metabolism / Q000648:ultrastructure; D033681:Mitochondrial Membrane Transport Proteins / Q000276:immunology*; D009928:Organ Specificity; D010101:Oxygen Consumption; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D033681:Mitochondrial Membrane Transport Proteins