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

Nature Protocols volume 3, pages 965–976 (2008)Cite this article

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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Figure 1: Scheme showing the principle of analysis of mitochondria in situ by selective plasma membrane permeabilization.
Figure 2: Example of a specific substrate inhibitor titration oxygraphic protocol for step-by-step analysis of various segments of the respiratory chain.
Figure 3: Example of isolation of rat m. soleus muscle fibers.
Figure 4
Figure 5
Figure 6: Examples of poor-quality preparations of permeabilized muscle fibers.

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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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Authors and Affiliations

  1. Department of General and Transplant Surgery, Daniel Swarovski Research Laboratory, 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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  1. Andrey V Kuznetsov
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Correspondence to Andrey V Kuznetsov.

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Kuznetsov, A., Veksler, V., Gellerich, F. et al. Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat Protoc 3, 965–976 (2008). https://doi.org/10.1038/nprot.2008.61

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