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| {{Abstract
| | #REDIRECT [[EBEC2014]] |
| |title=Combined high-resolution respirometry and fluorometry. Validation of safranin for determination of mitochondrial membrane potential.
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| |authors=Krumschnabel G, Eigentler A, Fasching M, Gnaiger E
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| |year=2014
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| |event=EBEC2014
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| |abstract=Mitochondrial membrane potential (mtMP) is closely intertwined with oxidative phosphorylation (OXPHOS). The exact nature of the interactions of respiration (flux) and mtMP (force) under various physiological and pathological conditions remains unclear, partially due to methodological limitations. We introduce the combination of high-resolution respirometry and fluorometry with the OROBOROS Oxygraph-2k, using the widely applied mtMP indicator safranin. OXPHOS analysis with mouse brain homogenate revealed that safranin inhibits Complex I linked OXPHOS capacity at commonly applied concentrations and targets primarily the phosphorylation system, without effect on LEAK respiration. Complex II linked OXPHOS capacity was inhibited by <20% at 2 µM safranin sufficient for mtMP monitoring. mtMP was higher in the LEAK state without adenylates (''L<sub>N</sub>'') than at identical LEAK respiration after ADP stimulation and inhibition by oligomycin (''L<sub>Omy</sub>''). Maximum ETS capacity was reached in uncoupler titrations before mtMP was fully collapsed, whereas respiration was inhibited at increasing uncoupler concentrations and further reduction of mtMP. Examining a pharmacologically induced state of Complex II dysfunction, mtMP was rather insensitive to 50% inhibition of OXPHOS, but responded strongly to addition of inhibitors when respiration was minimized by substrate depletion. The optimum uncoupler concentration supporting maximum ETS capacity varied as a function of pharmacological intervention. Taken together, combined measurement of respiration and mtMP greatly enhances the diagnostic potential of OXPHOS analysis. Respirometric validation of inhibitory and uncoupling effects is mandatory for any fluorophore applied for probing mtMP, in any respiratory state, type of tissue and pathophysiological condition.
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| Supported by K-Regio project ''[[MitoCom]]''.
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| |keywords=Safranin
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| |mipnetlab=AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS, AT Innsbruck MitoCom
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| }}
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| {{Labeling
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| |area=Respiration
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| |organism=Mouse
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| |tissues=Nervous system
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| |preparations=Homogenate
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| |topics=mt-Membrane potential
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| |couplingstates=LEAK, OXPHOS, ETS
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| |substratestates=CI, CII
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| |instruments=Oxygraph-2k, Fluorometry
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| }}
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