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

Nature Protocols volume 3, pages 965976 (2008) | Download Citation

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Abstract

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.

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Acknowledgements

This work was supported in part by a research grant from the Austrian Cancer Society/Tyrol to A.V.K. and by grants from Deutsche Forschungsgemeinschaft (KU-911/15-1, SCHR-562/4-3) and BMBF (01GZ0704) to W.S.K., by Agence National de la Recherche (project no. BLAN07-2_188128) France and by grants of Estonian Science Foundation (N° 6142 and 7117) to V.S. The authors thank Drs. J. Troppmair and A. Amberger for their insightful comments on this manuscript and their helpful discussions.

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Affiliations

  1. Daniel Swarovski Research Laboratory, Department of General and Transplant Surgery, Innsbruck Medical University, Innsbruck A6020, Austria.

    • Andrey V Kuznetsov
    •  & Raimund Margreiter
  2. INSERM U-769, Châtenay-Malabry F-92296, France.

    • Vladimir Veksler
  3. Université Paris Sud, Châtenay-Malabry F-92296, France.

    • Vladimir Veksler
  4. KeyNeurotek Pharmaceuticals AG, ZENIT Technology Park, Magdeburg D-39120, Germany.

    • Frank N Gellerich
  5. Laboratory of Bioenergetics, Joseph Fourier University, Grenoble F-38041, France.

    • Valdur Saks
  6. Department of Epileptology and Life & Brain Center, University of Bonn Medical Center, Bonn D-53105, Germany.

    • Wolfram S Kunz
  7. Institute of Biochemistry II, Friedrich-Schiller-University Jena, Jena D-07743, Germany.

    • Wolfram S Kunz

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Correspondence to Andrey V Kuznetsov.

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https://doi.org/10.1038/nprot.2008.61

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