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Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes

Nature Protocols volume 1, pages 2563–2572 (2006)Cite this article

Abstract

Mitochondrial dysfunction has been associated with a variety of currently marketed therapeutics and has also been implicated in many disease states. Alterations in the rate of oxygen consumption are an informative indicator of mitochondrial dysfunction, but the use of such assays has been limited by the constraints of traditional measurement approaches. Here, we present a high-throughput, fluorescence-based methodology for the analysis of mitochondrial oxygen consumption using a phosphorescent oxygen-sensitive probe, standard microtitre plates and plate reader detection. The protocol describes the isolation of mitochondria from animal tissue, initial establishment and optimization of the oxygen consumption assay, subsequent screening of compounds for mitochondrial toxicity (uncoupling and inhibition), data analysis and generation of dose-response curves. It allows dozens of compounds (or hundreds of assay points) to be analyzed in a single day, and can be further up-scaled, automated and adapted for other enzyme- and cell-based screening applications.

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Figure 1: Diagrammatic representation of measurement principles.
Figure 2: Recommended plate maps.
Figure 3: Sample data output for inhibition analysis showing intensity profiles (a), transformed linearized profiles (b) and inversed normalized slopes (c) showing the effect of increasing KCN concentrations on glutamate/malate-driven ADP-activated respiration.

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Acknowledgements

Parts of this work were supported by the Marine Institute and Marine RTDI Measure, Productive Sector Operational Programme, National Development Plan 2000–2006, Grant-aid agreement No AT-04-01-01, which is gratefully acknowledged.

Author information

Authors and Affiliations

  1. Pfizer Drug Safety Research & Development, San Diego, USA

    Yvonne Will

  2. Luxcel Biosciences Ltd., BioTransfer Unit, Suite 3.32, BioInnovation Centre, UCC, Cork, Ireland

    James Hynes

  3. Tyndal National Institute, Lee Maltings, Prospect Row, Cork, Ireland

    Vladimir I Ogurtsov

  4. Biochemistry Department, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, Ireland

    Dmitri B Papkovsky

Authors
  1. Yvonne Will
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  2. James Hynes
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  3. Vladimir I Ogurtsov
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  4. Dmitri B Papkovsky
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Corresponding author

Correspondence to Dmitri B Papkovsky.

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Competing interests

D. Papkovsky is one of the stakeholders of Luxcol Biosciences. All the other authors have no competing financial interests.

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Will, Y., Hynes, J., Ogurtsov, V. et al. Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes. Nat Protoc 1, 2563–2572 (2006). https://doi.org/10.1038/nprot.2006.351

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