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High-Throughput Analysis of Mitochondrial Oxygen Consumption

  • James Hynes
  • Rachel L. Swiss
  • Yvonne Will
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1782)

Abstract

Interest in the investigation of mitochondrial dysfunction has seen a resurgence over recent years due to the implication of such dysfunction in both drug-induced toxicity and a variety of disease states. Here we describe a methodology to assist in such investigations whereby the oxygen consumption of isolated mitochondria is assessed in a high-throughput fashion using a phosphorescent oxygen-sensitive probe, standard microtiter plates, and plate reader detection. The protocols provided describe the required isolation procedures, initial assay optimization, and subsequent compound screening. Typical data is also provided illustrating the expected activity levels as well as recommended plate maps and data analysis approaches.

Key words

Mitochondria OXPHOS Oxygen consumption Toxicity Respiration Polarimetry Oxygen-sensitive probes Toxicity Drug safety testing 

Supplementary material

427050_2_En_4_MOESM1_ESM.doc (71 kb)
Supplemental Data 1: Data Transformation and calculation of UC50 and IC50 data (DOC 3676 kb)

References

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    Dykens JA, Marroquin LD, Will Y (2007) Strategies to reduce late-stage drug attrition due to mitochondrial toxicity. Expert Rev Mol Diagn 7:161–175CrossRefPubMedGoogle Scholar
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    Wallace KB (2008) Mitochondrial off targets of drug therapy. Trends Pharmacol Sci 29:361–366CrossRefPubMedGoogle Scholar
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    Papkovsky DB, Hynes J, Will Y (2006) Respirometric screening technology for ADME-Tox studies. Expert Opin Drug Metab Toxicol 2:313–323CrossRefPubMedGoogle Scholar
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    Will Y, Hynes J, Ogurtsov VI, Papkovsky DB (2007) Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes. Nat Protoc 1:2563–2572CrossRefGoogle Scholar
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    Hynes J, Hill R, Papkovsky DB (2006) The use of a fluorescence-based oxygen uptake assay in the analysis of cytotoxicity. Toxicol In Vitro 20:785–792CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Luxcel BiosciencesCorkIreland
  2. 2.Pfizer Global R&D, Compound Safety Prediction-WWMC, Cell Based Assays and Mitochondrial BiologyGrotonUSA

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