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Difference between revisions of "MiPNet12.23 FiberRespiration"

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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.
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]]Mitochondrial respiration in permeabilized fibres: Needle biopsies from horse skeletal muscle.
|info=[[File:PDF.jpg|50px|link=http://wiki.oroboros.at/images/4/49/MiPNet12.23_FibreRespiration.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet12.23_FibreRespiration.pdf Versions]
|info=[[File:PDF.jpg|100px|link= http://wiki.oroboros.at/images/4/49/MiPNet12.23_FibreRespiration.pdf|Bioblast pdf]]  
|authors=OROBOROS
|authors=OROBOROS
|year=2014-11-07
|year=2014-11-07
|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, Gnaiger E (2014) Mitochondrial respiration in permeabilized fibres: Needle biopsies from horse skeletal muscle. Mitochondr Physiol Network 12.23(07):1-8.''' »[http://www.bioblast.at/index.php/File:MiPNet12.23_FibreRespiration.pdf Versions]


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).]
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).]

Revision as of 16:17, 8 March 2016

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O2k-Protocols contents
Mitochondrial respiration in permeabilized fibres: Needle biopsies from horse skeletal muscle.

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OROBOROS (2014-11-07) 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. »Versions

Methodological and conceptual features of highresolution respirometry are illustrated in an experiment with permeabilized fibres in the OROBOROS 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).

MiPNet12.23.jpg

Figure 1. Oxygen concentration ([μM] blue line) and oxygen flux per mg wet weight of muscle ([pmol∙s-1∙mg-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.


  1. LEAK state, L: Non-phosphorylating resting state with substrates for Complex I (glutamate+malate; GMN; without adenalytes, N).
  2. OXPHOS capacity with Complex I substrates, P: Respiratory capacity in the active coupled state (with ADP, GMD).
  3. 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).
  4. 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).
  5. 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.
  6. ETS capacity with CII substrate, E: ETS capacity with succinate, after blocking Complex I with rotenone.
  7. 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


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, O2k-Protocol 

O2k-Demo, O2k-Core 

Further information

  • 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. - Open Access
>> Excel Templates, DatLab Templates and DatLab-Demo Files