Wettmarshausen Jennifer, Perocchi Fabiana
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.
Methods Mol Biol. 2017;1567:15-32. doi: 10.1007/978-1-4939-6824-4_2.
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.
线粒体是许多细胞过程的核心场所，包括能量产生、细胞凋亡、离子稳态、铁和铜的处理、类固醇代谢、从头嘧啶合成以及血红素生物合成。对线粒体功能的研究通常需要纯化完整且具有呼吸功能的细胞器。在此，我们提供详细的方案，以从各种类型的哺乳动物细胞和小鼠组织中分离出粗提和纯品形式的功能性线粒体。我们介绍了使用氮气空化来破坏质膜并可重复分离高产率的富含线粒体的组分。通过这些程序分离出的线粒体是完整且偶联的，可直接用于多种下游分析，如氧气消耗和钙缓冲能力的测量。
