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.
Mitochondrial dysfunction is a common mechanism of drug-induced toxicity. Early identification of new chemical entities (NCEs) that perturb mitochondrial function is of significant importance to avoid attrition in later stages of drug development. One of the most informative ways of assessing mitochondrial dysfunction is by measuring mitochondrial oxygen consumption. However, the conventional polarographic method of measuring oxygen consumption is not amenable to high sample throughput or automation. We present an alternative, low-bulk, high-throughput approach to the analysis of isolated-mitochondrial oxygen consumption using luminescent oxygen-sensitive probes. These probes are dispensable and are analyzed in standard microtitre plates on a fluorescence plate reader. Respiratory substrate and adenosine diphosphate (ADP) dependencies of mitochondrial oxygen consumption were assessed using the fluorescence-based method, and results compared favourably to conventional polarographic analysis. To assess assay performance, the method was then applied to the analysis of a panel of classical modulators of oxidative phosphorylation. The effect of uncoupler concentration was analyzed in detail to identify factors which would be important in applying this method to large scale NCE screening and mechanistic investigations. Results demonstrate that the 96-well format can accommodate up to approximately 200 compounds/day at a single concentration or alternatively IC(50) values can be generated for approximately 25 compounds. Throughput may be increased by moving to a 384-well plate format.
I.; Taylor, Cormac T.; et al. Publication date 2010 Publication informationIntegrative Biology, 2 (9): Publisher The Royal Society of ChemistryItem record/more information http://hdl.handle.net/10197/5029Publisher's version (DOI) 10.1039/c0ib00021cDownloaded 2019-04-05T04:17:49ZThe UCD community has made this article openly available. Please share how this access benefits you. Your story matters! (@ucd_oa) Some rights reserved. For more information, please see the item record link above. Running title: Oxygen Gradients in Respiring SamplesAbstract Changes in cellular O 2 levels elicit adaptive responses which can lead to the activation of oxygen-dependent transcription factors such as the hypoxia-inducible factor (HIF). However, our understanding of the determinants of these processes is still incomplete. Using the new intracellular O 2 sensing technique, we monitored O 2 gradients in static cultures of adherent PC12 cells exposed to graded atmospheric O 2 at rest and upon metabolic stimulation. Under high atmospheric O 2 (10-21%) the respiration of resting cells dictated that local O 2 was moderately reduced, and at a certain threshold (6% in galactose medium) cell layer became practically anoxic. Furthermore, cell stimulation triggered a major redistribution of O 2 and a prominent 'hypoxic overshoot' effect mediated by diffusion. The deep, prolonged cell deoxygenation upon stimulation was matched by an increase in nuclear HIF-1α levels. In the presence of nitric oxide the hypoxic overshoot was truncated and HIF-1α stabilization inhibited. Thus, the main determinants which impact upon cellular O 2 levels and oxygen-sensitive signaling pathways are the atmospheric O 2 , sample geometry, cell density, respiration rate and its dynamics. Changes in any of these parameters can significantly alter the O 2 levels experienced by the cells and the subsequently activated signaling pathways. A model commonly used in in vitro studies is a static cell culture maintained at a constant external (atmospheric) pO 2 . For such a system, O 2 consumption rates and diffusion processes are important factors which contribute to O 2 transport to the cells from gaseous macro-phase 24,25 . Careful control of cellular O 2 in such experiments is still rare, and partial or complete deoxygenation and hypoxia-specific metabolic rearrangements may occur 17 and contribute to the observed biological effects. In this work, using quenchedphosphorescence O 2 sensing technique which allows real-time monitoring of both intra-and extracellular O 2 concentration 22,26,27 we investigated O 2 gradients in dense populations of neurosecretory pheochromocytoma PC12 cells cultured and differentiated under standard conditions in 96-well plates (dPC12). dPC12 cells possess active oxidative phosphorylation and glycolysis, produce robust responses to excitatory stimulation 22,26 , and they represent a convenient model for studies of brain function and pathologies 28 . For such a system with resting respiring dPC12 cells, steady state O 2 gradients at different...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.