Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Diphosphate / metabolism
  • Adenosine Triphosphatases / metabolism
  • Animals
  • Cell Membrane Permeability
  • Electron Transport
  • Humans
  • Mitochondria / metabolism
  • Mitochondria, Muscle / metabolism*
  • Muscle Fibers, Skeletal / metabolism*
  • Oxidative Phosphorylation
  • Rats


  • Adenosine Diphosphate
  • Adenosine Triphosphatases