MiPNet12.23 FiberRespiration: Difference between revisions
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|info=[[Media:MiPNet12.23 FibreRespiration.pdf|'''Open Access-MiPNet12.23.pdf''']]; [http://www.bioblast.at/index.php/File:MiPNet12.23_FibreRespiration.pdf Versions] | |info=[[Media:MiPNet12.23 FibreRespiration.pdf|'''Open Access-MiPNet12.23.pdf''']]; [http://www.bioblast.at/index.php/File:MiPNet12.23_FibreRespiration.pdf Versions] | ||
|authors=OROBOROS | |authors=OROBOROS | ||
|year= | |year=2011-12 | ||
|journal=Mitochondr Physiol Network | |journal=Mitochondr Physiol Network | ||
|abstract='''Lemieux H, Votion DM, Gnaiger E ( | |abstract='''Lemieux H, Votion DM, Gnaiger E (2011) Mitochondrial respiration in permeabilized fibres: Needle biopsies from horse skeletal muscle. Mitochondr Physiol Network 12.23(06).''' | ||
Methodological and conceptual features of highresolution respirometry are illustrated in an experiment with permeabilized fibres in the OROBOROS Oxygraph-2k (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).] | Methodological and conceptual features of highresolution respirometry are illustrated in an experiment with permeabilized fibres in the OROBOROS Oxygraph-2k (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).] | ||
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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). | #'''Residual oxygen consumption (ROX)''' due to oxidative side reactions in the permeabilized fibres, estimated after addition of Antimycin A (Inhibitor of Complex III). | ||
:>> Product: [http://www.oroboros.at/?oxygraph OROBOROS Oxygraph-2k], [[O2k-Catalogue_OROBOROS| O2k-Catalogue]] | :>> Product: [http://www.oroboros.at/?oxygraph OROBOROS Oxygraph-2k], [[O2k-Catalogue_OROBOROS| O2k-Catalogue]] | ||
|mipnetlab=AT_Innsbruck_OROBOROS, BE_Liege_Votion DM}} | |mipnetlab=AT_Innsbruck_OROBOROS, BE_Liege_Votion DM | ||
}} | |||
{{Labeling | {{Labeling | ||
|area=Respiration, Instruments;methods | |area=Respiration, Instruments;methods | ||
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|substratestates=CI, CII, CI+II | |substratestates=CI, CII, CI+II | ||
|instruments=Oxygraph-2k, Protocol | |instruments=Oxygraph-2k, Protocol | ||
|additional=O2k-Demo, O2k-Core}} | |additional=O2k-Demo, O2k-Core | ||
}} | |||
== Further information == | == Further information == | ||
:>> '''[http://www.oroboros.at/index.php?id=protocols_muscle_horse Excel Templates, DatLab Templates and DatLab-Demo Files]''' | :>> '''[http://www.oroboros.at/index.php?id=protocols_muscle_horse Excel Templates, DatLab Templates and DatLab-Demo Files]''' |
Revision as of 19:22, 14 June 2014
Mitochondrial respiration in permeabilized fibres: Needle biopsies from horse skeletal muscle. |
ยป Open Access-MiPNet12.23.pdf; Versions
OROBOROS (2011-12) Mitochondr Physiol Network
Abstract: Lemieux H, Votion DM, Gnaiger E (2011) Mitochondrial respiration in permeabilized fibres: Needle biopsies from horse skeletal muscle. Mitochondr Physiol Network 12.23(06).
Methodological and conceptual features of highresolution respirometry are illustrated in an experiment with permeabilized fibres in the OROBOROS Oxygraph-2k (O2k). The experiment demonstrates manual titrations applied to study respiratory capacity in permeabilized fibres. Application of the 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 (IOC44; Schroecken, Austria).
Figure 1. Oxygen concentration ([ฮผM] blue line) and oxygen flux per mg wet weight of muscle ([pmolโs-1โmg-1] red lines) in O2k chamber B, in permeabilized fibres from horse skeletal muscle with the standard titration protocol.
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.
- LEAK state, L: Non-phosphorylating resting state with substrates for Complex I (glutamate+malate; GMN; without adenalytes, N).
- OXPHOS capacity with Complex I substrates, P: Respiratory capacity in the active coupled state (with ADP, GMD).
- Cytochrome c test for quality control: 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).
- OXPHOS capacity with Complex I+II substrates, P: 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).
- Electron transfer system (ETS) capacity with CI+II substrates, E: 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.
- ETS capacity with CII substrate, E: ETS capacity with succinate, after blocking Complex I with rotenone.
- Residual oxygen consumption (ROX) due to oxidative side reactions in the permeabilized fibres, estimated after addition of Antimycin A (Inhibitor of Complex III).
- >> Product: OROBOROS Oxygraph-2k, O2k-Catalogue
โข O2k-Network Lab: AT_Innsbruck_OROBOROS, BE_Liege_Votion DM
Labels: MiParea: Respiration, Instruments;methods
Organism: Horse
Tissue;cell: Skeletal muscle
Preparation: Permeabilized tissue
Regulation: Coupling efficiency;uncoupling, Cyt c Coupling state: LEAK, OXPHOS, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property.
HRR: Oxygraph-2k, Protocol"Protocol" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property.
O2k-Demo, O2k-Core