Kuznetsov Andrey V, Veksler Vladimir, Gellerich Frank N, Saks Valdur, Margreiter Raimund, Kunz Wolfram S
Daniel Swarovski Research Laboratory, Department of General and Transplant Surgery, Innsbruck Medical University, Innsbruck A6020, Austria.
Nat Protoc. 2008;3(6):965-76. doi: 10.1038/nprot.2008.61.
Analysis of mitochondrial function is central to the study of intracellular energy metabolism, mechanisms of cell death and pathophysiology of a variety of human diseases, including myopathies, neurodegenerative diseases and cancer. However, important properties of mitochondria differ in vivo and in vitro. Here, we describe a protocol for the analysis of functional mitochondria in situ, without the isolation of organelles, in selectively permeabilized cells or muscle fibers using digitonin or saponin. A specially designed substrate/inhibitor titration approach allows the step-by-step analysis of several mitochondrial complexes. This protocol allows the detailed characterization of functional mitochondria in their normal intracellular position and assembly, preserving essential interactions with other organelles. As only a small amount of tissue is required for analysis, the protocol can be used in diagnostic settings in clinical studies. The permeabilization procedure and specific titration analysis can be completed in 2 h.
线粒体功能分析是细胞内能量代谢、细胞死亡机制以及多种人类疾病（包括肌病、神经退行性疾病和癌症）病理生理学研究的核心。然而，线粒体的重要特性在体内和体外有所不同。在此，我们描述了一种在原位分析功能性线粒体的方法，无需分离细胞器，在使用洋地黄皂苷或皂角苷选择性通透的细胞或肌肉纤维中进行。一种专门设计的底物/抑制剂滴定方法允许对几种线粒体复合物进行逐步分析。该方法能够在正常细胞内位置和组装状态下详细表征功能性线粒体，保留与其他细胞器的重要相互作用。由于分析仅需少量组织，该方法可用于临床研究的诊断环境。通透过程和特定滴定分析可在2小时内完成。
