Protocol abstract


Nature Protocols 1, 2563 - 2572 (2007)
Published online: 4 January 2007 | doi:10.1038/nprot.2006.351

Subject Category: Isolation, purification and separation

Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes

Yvonne Will1, James Hynes2, Vladimir I Ogurtsov3 & Dmitri B Papkovsky4


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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  1. Pfizer Drug Safety Research & Development, San Diego, USA.
  2. Luxcel Biosciences Ltd., BioTransfer Unit, Suite 3.32, BioInnovation Centre, UCC, Cork, Ireland.
  3. Tyndal National Institute, Lee Maltings, Prospect Row, Cork, Ireland.
  4. Biochemistry Department, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, Ireland.

Correspondence to: Dmitri B Papkovsky4 e-mail: d.papkovsky@ucc.ie

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