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| {{Publication | | {{Publication |
| |title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]]Mitochondrial respiration in permeabilized fibres: Needle biopsies from horse skeletal muscle. [[Media:MiPNet12.23 FibreRespiration.pdf|Β»Bioblast pdfΒ«]] | | Β |
| |info=[http://www.bioblast.at/index.php/File:MiPNet12.23_FibreRespiration.pdf Versions]
| | |title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]]Mitochondrial respiration in permeabilized fibers: Needle biopsies from horse skeletal muscle. |
| |authors=OROBOROS | | |info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/4/49/MiPNet12.23_FibreRespiration.pdf|Bioblast pdf]] Β»[http://www.bioblast.at/index.php/File:MiPNet12.23_FibreRespiration.pdf Versions] |
| |year=2014-11-07 | | |authors=Oroboros |
| | |year=2019-01-10 |
| |journal=Mitochondr Physiol Network | | |journal=Mitochondr Physiol Network |
| |abstract=Lemieux H, Votion DM, Gnaiger E (2014) Mitochondrial respiration in permeabilized fibres: Needle biopsies from horse skeletal muscle. Mitochondr Physiol Network 12.23(07):1-8. | | |abstract=Lemieux H, Votion DM, Doerrier C, Gnaiger E (2019) Mitochondrial respiration in permeabilized fibers: Needle biopsies from horse skeletal muscle. Mitochondr Physiol Network 12.23(08):1-4. |
|  | | :» O2k-Protocols:[[SUIT-011| SUIT-011]] |
| Methodological and conceptual features of highresolution respirometry are illustrated in an experiment with permeabilized fibres in the OROBOROS O2k. The experiment demonstrates [http://bioblast.at/images/e/ef/MiPNet09.12_O2k-Titrations.pdf manual titrations] applied to study respiratory capacity in permeabilized fibres. Application of the [http://www.oroboros.at/?datlab DatLab 4 software] is shown for on-line data analysis [[MiPNet12.09]] The following guideline describes the experimental protocol and includes a short discussion of results. The experiments was carried out by participants of an O2k-Course on HRR in December 2007 [http://www.oroboros.at/index.php?id=ioc44 (IOC44; Schroecken, Austria).]
| | :Β» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] |
| Β | | |mipnetlab=AT_Innsbruck_Oroboros, BE_Liege_Votion DM |
| [[Image:MiPNet12.23.jpg|center|100px|frame]]
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| Figure 1. Oxygen concentration ([ΞΌM] blue line) and oxygen flux per mg wet weight of muscle ([pmolβs-1βmg-1] red lines) in O2k chamber B, in permeabilized fibres from horse skeletal muscle with the standard titration protocol.
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| A multiple substrate-uncoupler-inhibitor titration protocol (Fig. 1) was developed for respiratory studies of permeabilized muscle fibres. The experiment was aimed at an assessment of a sequence of titrations, inducing stepwise defined respiratory states.
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| #<span style="color:red"> '''LEAK state, ''L'':'''</span> Non-phosphorylating resting state with substrates for Complex I (glutamate+malate; GMN; without adenalytes, N).
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| #<span style="color:green">'''OXPHOS capacity with Complex I substrates, ''P'':'''</span> Respiratory capacity in the active coupled state (with ADP, GMD).
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| #<span style="color:black">'''Cytochrome ''c'' test for quality control:'''</span> Further addition of cytochrome ''c'' yields a test for integrity of the outer mitochondrial membrane (loss of cytochrome c would be indicated by a stimulation of respiration).
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| #<span style="color:green">'''OXPHOS capacity with Complex I&II substrates, ''P'':'''</span> Respiratory stimulation by convergent electron flow from Complexes I&II at the Q-junction, in the coupled state after further addition of succinate, as an estimate of OXPHOS capacity (Gnaiger 2009).
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| #<span style="color:blue">'''Electron transfer system (ETS) capacity with CI&II substrates, ''E'':'''</span> Uncoupling by FCCP titration (avoiding inhibition by high FCCP concentrations), as a test for limitation of OXPHOS relative to ETS capacity by the phosphorylation system.
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| #<span style="color:blue">'''ETS capacity with CII substrate, ''E'':'''</span> ETS capacity with succinate, after blocking Complex I with rotenone.
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| #'''Residual oxygen consumption (ROX)''' due to oxidative side reactions in the permeabilized fibres, estimated after addition of Antimycin A (Inhibitor of Complex III).
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| :>> Product: [http://www.oroboros.at/?oxygraph OROBOROS Oxygraph-2k], [[O2k-Catalogue_OROBOROS| O2k-Catalogue]] | |
| |mipnetlab=AT_Innsbruck_OROBOROS, BE_Liege_Votion DM | |
| }}
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| {{Labeling
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| |area=Respiration, Instruments;methods
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| |organism=Horse
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| |tissues=Skeletal muscle
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| |preparations=Permeabilized tissue
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| |topics=Coupling efficiency;uncoupling, Cyt c
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| |couplingstates=LEAK, OXPHOS, ETS
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| |substratestates=CI, CII, CI&II
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| |instruments=Oxygraph-2k, Protocol
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| |additional=O2k-Demo, O2k-Core
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| }} | | }} |
| == Further information ==
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| * Votion DM, Gnaiger E, Lemieux H, Mouithys-Mickalad A, Serteyn D (2012) Physical fitness and mitochondrial respiratory capacity in horse skeletal muscle. PLoS One 7: e34890. - [[Votion 2012 PLoS One |Open Access]]
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| :>> '''[http://www.oroboros.at/?protocols_muscle_horse Excel Templates, DatLab Templates and DatLab-Demo Files]'''
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