Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes

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.

[1]  J. Paul Robinson,et al.  Mitochondrial Complex I Inhibitor Rotenone Induces Apoptosis through Enhancing Mitochondrial Reactive Oxygen Species Production* , 2003, The Journal of Biological Chemistry.

[2]  Dmitri B Papkovsky,et al.  The use of a fluorescence-based oxygen uptake assay in the analysis of cytotoxicity. , 2006, Toxicology in vitro : an international journal published in association with BIBRA.

[3]  F. Levi-Schaffer,et al.  Role of reactive oxygen species (ROS) in apoptosis induction , 2000, Apoptosis.

[4]  J. Lakowicz Principles of fluorescence spectroscopy , 1983 .

[5]  R. Lapidus,et al.  Spermine inhibition of the permeability transition of isolated rat liver mitochondria: an investigation of mechanism. , 1993, Archives of biochemistry and biophysics.

[6]  S. Ballinger Mitochondrial dysfunction in cardiovascular disease. , 2005, Free radical biology & medicine.

[7]  Dmitri B Papkovsky,et al.  Investigation of drug-induced mitochondrial toxicity using fluorescence-based oxygen-sensitive probes. , 2006, Toxicological sciences : an official journal of the Society of Toxicology.

[8]  Elmar Heinzle,et al.  On-line oxygen uptake rate and culture viability measurement of animal cell culture using microplates with integrated oxygen sensors , 2004, Biotechnology Letters.

[9]  Induction , 1999 .

[10]  D. Buckley,et al.  A cell viability assay based on monitoring respiration by optical oxygen sensing. , 2000, Analytical biochemistry.

[11]  K. Wallace,et al.  Doxorubicin-induced cardiac mitochondrionopathy. , 2003, Pharmacology & toxicology.

[12]  Ingo Klimant,et al.  Gas sensing in microplates with optodes: influence of oxygen exchange between sample, air, and plate material. , 2005, Biotechnology and bioengineering.

[13]  B. Day,et al.  Mitochondrial toxicity of nrti antiviral drugs: an integrated cellular perspective , 2003, Nature Reviews Drug Discovery.

[14]  Christian Krause,et al.  Microplates with integrated oxygen sensors for kinetic cell respiration measurement and cytotoxicity testing in primary and secondary cell lines. , 2005, Assay and drug development technologies.

[15]  Dmitri B. Papkovsky,et al.  Phosphorescent oxygen-sensitive materials for biological applications , 2005 .

[16]  Dmitri B Papkovsky,et al.  Respirometric Screening Technology for ADME-Tox studies , 2006, Expert opinion on drug metabolism & toxicology.

[17]  Michael Roden,et al.  Fenofibrate Impairs Rat Mitochondrial Function by Inhibition of Respiratory Complex I , 2004, Journal of Pharmacology and Experimental Therapeutics.

[18]  D. Amacher Drug-associated mitochondrial toxicity and its detection. , 2005, Current medicinal chemistry.

[19]  G. Moyle Mechanisms of HIV and Nucleoside Reverse Transcriptase Inhibitor Injury to Mitochondria , 2005, Antiviral therapy.

[20]  R C Scaduto,et al.  Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. , 1999, Biophysical journal.

[21]  D. Green,et al.  And all of a sudden it's over: mitochondrial outer-membrane permeabilization in apoptosis. , 2002, Biochimie.

[22]  M. Mattson,et al.  Mitochondria in cell death: novel targets for neuroprotection and cardioprotection. , 2003, Trends in molecular medicine.

[23]  J. Demas,et al.  Oxygen sensors based on luminescence quenching. , 1999, Analytical chemistry.

[24]  L. B. Chen,et al.  Mitochondrial membrane potential monitored by JC-1 dye. , 1995, Methods in enzymology.

[25]  Dmitri B Papkovsky,et al.  Methods in optical oxygen sensing: protocols and critical analyses. , 2004, Methods in enzymology.

[26]  J. Bruce Pitner,et al.  Novel Fluorescent Technology Platform for High Throughput Cytotoxicity and Proliferation Assays , 2000, Journal of biomolecular screening.

[27]  Division on Earth Guide for the Care and Use of Laboratory Animals , 1996 .

[28]  Maile R. Brown,et al.  Mitochondrial aging and dysfunction in Alzheimer's disease , 2005, Progress in Neuro-Psychopharmacology and Biological Psychiatry.

[29]  M. Crompton,et al.  The mitochondrial permeability transition pore. , 1999, Biochemical Society symposium.

[30]  P. Sirvent,et al.  Simvastatin induces impairment in skeletal muscle while heart is protected. , 2005, Biochemical and biophysical research communications.

[31]  Dmitri B. Papkovsky,et al.  Rapid High-Throughput Assessment of Aerobic Bacteria in Complex Samples by Fluorescence-Based Oxygen Respirometry , 2006, Applied and Environmental Microbiology.

[32]  Dmitri B. Papkovsky,et al.  Fluorescence-Based Cell Viability Screening Assays Using Water-Soluble Oxygen Probes , 2003, Journal of biomolecular screening.

[33]  W. Willis,et al.  Pyruvate and citric acid cycle carbon requirements in isolated skeletal muscle mitochondria. , 2004, American journal of physiology. Cell physiology.

[34]  L. Kangas,et al.  Bioluminescence of cellular ATP: a new method for evaluating cytotoxic agents in vitro. , 1984, Medical biology.

[35]  Dmitri B Papkovsky,et al.  Fluorescence based oxygen uptake analysis in the study of metabolic responses to apoptosis induction. , 2005, Journal of immunological methods.

[36]  M. V. Heiden,et al.  Outer mitochondrial membrane permeability can regulate coupled respiration and cell survival. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[37]  John Alderman,et al.  A low-volume platform for cell-respirometric screening based on quenched-luminescence oxygen sensing. , 2004, Biosensors & bioelectronics.

[38]  Benjamin A. DeGraff,et al.  Peer Reviewed: Oxygen Sensors Based on Luminescence Quenching. , 1999 .

[39]  Ingo Klimant,et al.  Integrated optical sensing of dissolved oxygen in microtiter plates: A novel tool for microbial cultivation , 2003, Biotechnology and bioengineering.

[40]  John Davenport,et al.  Optical oxygen microrespirometry as a platform for environmental toxicology and animal model studies. , 2005, Environmental science & technology.