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