Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells.
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.
Kuznetsov
Andrey V
AV
Daniel Swarovski Research Laboratory, Department of General and Transplant Surgery, Innsbruck Medical University, Innsbruck A6020, Austria. andrey.kuznetsov@uki.at
Veksler
Vladimir
V
Gellerich
Frank N
FN
Saks
Valdur
V
Margreiter
Raimund
R
Kunz
Wolfram S
WS
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002463:Cell Membrane Permeability; D004579:Electron Transport; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D008931:Mitochondria, Muscle / Q000378:metabolism*; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D010085:Oxidative Phosphorylation; D051381:Rats
D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases