title: Isolation of Functional Mitochondria from Cultured Cells and Mouse Tissues.
abstract: Mitochondria serve as the center stage for a number of cellular processes, including energy production, apoptosis, ion homeostasis, iron and copper processing, steroid metabolism, de novo pyrimidine, and heme biosynthesis. The study of mitochondrial function often requires the purification of intact and respiratory-competent organelles. Here, we provide detailed protocols to isolate functional mitochondria from various types of mammalian cells and mouse tissues, in both crude and pure forms. We introduce the use of nitrogen cavitation for the disruption of plasma membrane and the reproducible isolation of mitochondria-enriched fractions of high yield. Mitochondria that are isolated by these procedures are intact and coupled and can directly be used for several downstream analyses, such as measurements of oxygen consumption and calcium buffering capacity.
authors.affiliation: Genzentrum, Department of Biochemistry, Ludwig-Maximilians University, Feodor-Lynen Strasse 25, 81377, Munich, Germany.|Institute for Diabetes and Obesity, Helmholtz Zentrum München, 85764, Neuherberg, Germany.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Wettmarshausen
authors.affiliation: Gene Center, LMU Munich and Institute for Diabetes and Obesity, Helmholtz Zentrum München, Munich, Germany. perocchi@genzentrum.lmu.de.
authors.forename: Fabiana
authors.initials: F
authors.lastname: Perocchi
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods*; D002460:Cell Line; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient / Q000379:methods; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D008931:Mitochondria, Muscle / Q000378:metabolism; D057188:Workflow
chemical_list: D002118:Calcium
keywords: Crude mitochondria; Mitochondrial integrity; Nitrogen cavitation; Organelle isolation; Pure mitochondria