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Difference between revisions of "IOC recommended reading"

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{{Mainpage OROBOROS}}
{{OROBOROS header page name}}
[[File:MITOEAGLE-representation.jpg|right|180px|link=http://www.mitoglobal.org/index.php/COST Action MitoEAGLE|COST Action MitoEAGLE]]


== <big><big>HRR:</big></big> High-resolution respirometry ==
__TOC__


Gnaiger E (2014) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 4th ed. Mitochondr Physiol Network 19.12. OROBOROS MiPNet Publications, Innsbruck:80 pp. »[[Gnaiger 2014 MitoPathways |Open Access]]«
== MitoEAGLE 'Mitochondrial physiology' BEC 2020.1 ==
:* The Blue Book: Background on OXPHOS analysis.
:::* <big>Gnaiger Erich et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1. doi:10.26124/bec:2020-0001.v1. - [[BEC_2020.1_doi10.26124bec2020-0001.v1 |»Bioblast link«]]</big>
<br />


[[Image:O2k-Workshops.png|right|180px|link=OROBOROS_Events]]
[[Image:O2k-Publications.jpg|left|80px|link=O2k-Publications: Topics|O2k-Publications in the MiPMap]]


Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley:327-52. »[[Gnaiger_2008_POS |Bioblast Access]]«
:* Methodological introduction, comparison of HRR with traditional oxygraphy.
:* Dramatically different: high-resolution respirometry - the OROBOROS [[Oxygraph-2k]].
:* Calibration of polarographic oxygen sensors and oxygen concentration in respiration media at air saturation.
:* From Oxygraph slopes to respiratory flux corrected for background effects.
:* Phosphorylation or coupling control protocol (PCP, CCP) with intact cells.
:* Titration steps of the CCP.
:* Experimental example.
:* Flux control ratios from the CCP.
:* Intact cells, permeabilized cells and tissue, or isolated mitochondria?


== Workshop material==


Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128:277-97. »[[Gnaiger 2001 Respir Physiol |Link]]« – A detailed introduction into high-resolution respirometry with particular emphasis on kinetics and measurements at low oxygen.  
:::* Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibres from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782 (Palmeira CM, Moreno AJ, eds): Mitochondrial Bioenergetics, 978-1-4939-7830-4. - [[Doerrier 2018 Methods Mol Biol |»Bioblast link«]]
:* Mitochondrial kinetics measured by high-resolution respirometry  
:::* Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. Bioenerg Commun 2020.2. - [[Gnaiger 2020 BEC MitoPathways |»Bioblast link«]] - ''A new edition with updated terminology and more ..''
:* Calibrations and corrections for response time and instrumental background
:::* Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen  peroxide fluxes in permeabilized cells, tissue homogenate and isolated  mitochondria. Biomolecules 5:1319-38. [[Makrecka-Kuka 2015 Biomolecules|»Bioblast pdf«]]
:* Steady-state injection respirometry
:::* Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81. - [[Krumschnabel 2014 Methods Enzymol |»Bioblast link«]]
:* Mitochondrial respiratory control at low oxygen
:::* [[Media:MiPNet22.11_O2k-FluoRespirometer-manual.pdf|'''O2k-Manual.pdf''']]: O2k-Manual for [[DatLab 7]].
:* Apparent oxygen affinity and catalytic efficiency of mitochondrial respiration
:* Effect of ADP and oxygen limitation on ADP/O2 flux ratios
:* The low-oxygen environment of the cell: Mitochondria between hypoxic and oxidative stress






== HRR: high-resolution respirometry and SUIT protocols ==


[[Image:O2k-Publications.jpg|left|50px|link=O2k-Publications: Topics|O2k-Publications in the MiPMap]]
== SUIT protocols for high-resolution respirometry ==


:::* <big>Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibres from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782 (Palmeira CM, Moreno AJ, eds): Mitochondrial Bioenergetics, 978-1-4939-7830-4. - [[Doerrier 2018 Methods Mol Biol |»Bioblast link«]]</big>


Gnaiger E (2014) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 4th ed. Mitochondr Physiol Network 19.12. OROBOROS MiPNet Publications, Innsbruck:80 pp. »[[Gnaiger 2014 MitoPathways |Open Access]]«
:::* <big>Lemieux H, Blier PU, Gnaiger E (2017) Remodeling pathway control of mitochondrial respiratory capacity by temperature in mouse heart: electron flow through the Q-junction in permeabilized fibers. Sci Rep 7:2840, DOI:10.1038/s41598-017-02789-8. - [[Lemieux 2017 Sci Rep |»Bioblast link«]]</big>
:* The Blue Book: OXPHOS analysis; CI-linked, CII-linked, CI<small>&</small>II-linked pathways, respiratory states, abbreviations.
::::» Updated terminology with SUIT protocols.
::::» NADH-linked or N-pathways (CI-entry into Q); succinate-linked or S-pathway (CII-entry into Q); NS-pathway (convergent CI<small>&</small>II-entry into Q).  


:::* <big>Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. BEC 2020.2. - [[Gnaiger 2020 BEC MitoPathways |»Bioblast link«]]</big>
::::» ''A hard copy of this monograph on mitochondrial physiology will be included in the workshop material. The Blue Book is available on-line free of charge and provides a background on OXPHOS analysis'':
::::» From oxygen concentration to oxygen flux '''••''' Membrane potential: electric and chemical forces '''••''' Coupling and efficiency '''••''' Biochemical thresholds
::::» Respiratory states
::::» MitoPathways - the electron transfer-pathway
::::» Normalization of mitochondrial respiration: coupling control and pathway control.


Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810: 25-58. »[[Pesta 2012 Methods Mol Biol |Bioblast Access]]«
:* Introduction to high-resolution respirometry (HRR), SUIT protocols and flux control ratios.


:::* <big>Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley:327-52. - [[Gnaiger_2008_POS |»Bioblast link«]]</big>
::::» Methodological introduction, comparison of HRR with traditional oxygraphy.
::::» Dramatically different: high-resolution respirometry - the [[Oroboros O2k]].
::::» Calibration of polarographic oxygen sensors and oxygen concentration in respiration media at air saturation.
::::» From Oxygraph slopes to respiratory flux corrected for background effects.
::::» Phosphorylation or coupling control protocol (PCP, CCP) with intact cells.
::::» Titration steps of the CCP.
::::» Experimental example.
::::» Flux control ratios from the CCP.
::::» Living cells, permeabilized cells and tissue, or isolated mitochondria?


» '''[[MitoPedia: Respiratory states]]'''
[[Image:P.jpg|link=OXPHOS capacity|OXPHOS]] [[Image:R.jpg|link=ROUTINE respiration|ROUTINE]] [[Image:E.jpg|link=ETS capacity|ETS]] [[Image:L.jpg|link=LEAK respiration|LEAK]] [[Image:ROX.jpg|link=Residual oxygen consumption|ROX]]




[[Image:O2k-Fluorometer.JPG|60px|left|link=O2k-Fluorometer|O2k-Fluorometer]]
== O2k-Fluorometry ==


* [[MiPNet17.05 O2k-Fluorescence LED2-Module |»Manual: '''O2k-Fluorometry''']]
:::* '''[[MitoPedia: SUIT]]'''
* Krumschnabel et al (2014) Methods Enzymol. »[[Krumschnabel 2014 Methods Enzymol |Link]]«
 
* Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) O2k-Fluorometry: HRR and simultaneous determination of mt membrane potential with safranin. Mitochondr Physiol Network 19.19(01):1-5. »[[MiPNet19.19_Safranin_Data_Acquisition_and_Analysis |Open Access]]«
 
* Krumschnabel G, Makrecka-Kuka M, Kumphune S, Fontana-Ayoub M, Fasching M, Gnaiger E (2014) O2k-Fluorometry: HRR and simultaneous determination of H2O2 production with Amplex Red. Mitochondr Physiol Network 19.20(01):1-6. »[[MiPNet19.20_Amplex_Red_Data_Acquisition_and_Analysis |Open Access]]«
:::* '''[[MitoPedia: Respiratory states]]'''
* Eigentler A, Fontana-Ayoub M, Gnaiger E (2013) O2k-Fluorometry: HRR and H<sub>2</sub>O<sub>2</Sub> production in mouse cardiac tissue homogenate. Mitochondr Physiol Network 18.05(01): 1-6. »[[MiPNet18.05 Amplex-Mouse-heart |Open Access]]«
[[Image:P.jpg|link=OXPHOS capacity|OXPHOS]] [[Image:R.jpg|link=ROUTINE respiration|ROUTINE]] [[Image:E.jpg|link=ET capacity|ETS]] [[Image:L.jpg|link=LEAK respiration|LEAK]] [[Image:ROX.jpg|link=Residual oxygen consumption|ROX]]
* '''[[O2k-Publications: O2k-Fluorometry]]'''
 
 
[[Image:O2k-Fluorometer.JPG|60px|left|link=O2k-Fluorometer|O2k-FluoRespirometer]]
== O2k-FluoRespirometry ==
 
:::* Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution  respirometry for simultaneous measurement of oxygen and hydrogen  peroxide fluxes in permeabilized cells, tissue homogenate and isolated  mitochondria. Biomolecules 5:1319-38. [[Makrecka-Kuka 2015 Biomolecules|»Bioblast pdf«]]
 
:::* Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81. - [[Krumschnabel 2014 Methods Enzymol |»Bioblast link«]]
 
:::* Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61. - [[Krumschnabel 2015 Methods Mol Biol |»Bioblast link«]]
 
:::* Eigentler A, Fontana-Ayoub M, Gnaiger E (2013) O2k-Fluorometry: HRR and H<sub>2</sub>O<sub>2</Sub> production in mouse cardiac tissue homogenate. Mitochondr Physiol Network 18.05(01):1-6. - [[MiPNet18.05 Amplex-Mouse-heart |»Bioblast link«]]
 
:::* '''[[O2k-Publications: O2k-Fluorometry]]'''




== TPP/ISE ==
== TPP/ISE ==


* O2k-MultiSensor System with ion selective electrodes (ISE) '''[[MiPNet15.03]]'''
:::* O2k-MultiSensor System with ion selective electrodes (ISE) '''[[MiPNet15.03 O2k-MultiSensor-ISE |MiPNet15.03]]'''
* Determination of membrane potential using TPP+ and an ion selective electrode (ISE) '''[[MiPNet14.05]]'''
 
* '''[[Calculation of mitochondrial membrane potential from measurements with a TPP electrode]]'''
:::* Determination of membrane potential using TPP+ and an ion selective electrode (ISE) '''[[MiPNet14.05]]'''
* Rottenberg H (1984) Membrane potential and surface potential in mitochondria: uptake and binding of lipophilic cations. J Membr Biol 81:127-38.
 
:::* Rottenberg H (1984) Membrane potential and surface potential in mitochondria: uptake and binding of lipophilic cations. J Membr Biol 81:127-38.




Line 69: Line 86:
== Further reading ==
== Further reading ==


Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41: 1837-1845. »[[Gnaiger 2009 Int J Biochem Cell Biol |Link]]«
:::* Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41:1837-45. - [[Gnaiger 2009 Int J Biochem Cell Biol |»Bioblast link«]]
:* Respirometry with permeabilized fibres and isolated mitochondria.
::::» Respirometry with permeabilized fibres and isolated mitochondria.
:* Convergent CI<small>&</small>II electron input and OXPHOS capacity.
::::» Convergent NS (CI<small>&</small>II) electron input and OXPHOS capacity.
:* Tissue-OXPHOS capacity in human permeabilized muscle fibres and isolated mitochondria.
::::» Tissue-OXPHOS capacity in human permeabilized muscle fibres and isolated mitochondria.
:* Tissue-OXPHOS capacity and functional diversity.
::::» Tissue-OXPHOS capacity and functional diversity.
 
 
:::* Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopsies of human muscle. Methods Mol Biol 810:25-58. - [[Pesta 2012 Methods Mol Biol |»Bioblast link«]]
::::» Introduction to high-resolution respirometry (HRR), SUIT protocols and flux control ratios.
 


:::* Scandurra FM, Gnaiger E (2010) Cell respiration under hypoxia: Facts and artefacts in mitochondrial oxygen kinetics. Adv Exp Med Biol 662:7-25. - [[Scandurra 2010 Adv Exp Med Biol |»Bioblast link«]]
::::» Isolated mitochondria and intact cells.
::::» Nanomolar resolution of oxygen concentration.
::::» Effects of cell density and activity.
::::» Closed versus open systems.
::::» ''p''<sub>50</sub> in vivo.


Scandurra FM, Gnaiger E (2010) Cell respiration under hypoxia: Facts and artefacts in mitochondrial oxygen kinetics. Adv Exp Med Biol 662: 7-25. »[[Scandurra 2010 Adv Exp Med Biol |Open Access]]«
:* Isolated mitochondria and intact cells.
:* Nanomolar resolution of oxygen concentration.
:* Effects of cell density and activity.
:* Closed versus open systems.
:* ''p''<sub>50</sub> in vivo.


:::* Gnaiger E, Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Steurer W, Margreiter R (2000) Mitochondria in the cold. In: Life in the Cold (Heldmaier G, Klingenspor M, eds) Springer, Heidelberg, Berlin, New York:431-42. - [[Gnaiger 2000 Life in the Cold |»Bioblast link«]]
::::» Isolated mitochondria and permeabilized muscle fibers.
::::» Optimization of mitochondrial cold storage • Mitochondrial respiration medium, [[MiR05]] • Mitochondrial cold ischemia-reperfusion injury.


Gnaiger E, Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Steurer W, Margreiter R (2000) Mitochondria in the cold. In: Life in the Cold (Heldmaier G, Klingenspor M, eds) Springer, Heidelberg, Berlin, New York:431-42. »[[Gnaiger 2000 Life in the Cold |Open Access]]«
:* Isolated mitochondria and permeabilized muscle fibers.
:* Optimization of mitochondrial cold storage • Mitochondrial respiration medium, [[MiR05]] • Mitochondrial cold ischemia-reperfusion injury.


:::* Renner K, Amberger A, Konwalinka G, Kofler R, Gnaiger E (2003) Changes of mitochondrial respiration, mitochondrial content and cell size after induction of apoptosis in leukemia cells. Biochim Biophys Acta 1642:115-23. - [[Renner 2003 Biochim Biophys Acta |»Bioblast link«]]
::::» Intact cells.
::::» Cytochrome ''c'' oxidase.
::::» Cytochrome ''c'' test.
::::» Respiration per million cells, per citrate synthase, per mg protein, or per cytochrome ''c'' oxidase activity.


Renner K, Amberger A, Konwalinka G, Kofler R, Gnaiger E (2003) Changes of mitochondrial respiration, mitochondrial content and cell size after induction of apoptosis in leukemia cells. Biochim Biophys Acta 1642:115-23. »[[Renner 2003 Biochim Biophys Acta |'''Link''']]«
:* Intact cells.
:* Cytochrome ''c'' oxidase.
:* Cytochrome ''c'' test.
:* Respiration per million cells, per citrate synthase, per mg protein, or per cytochrome ''c'' oxidase activity.


:::* Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128:277-97. - [[Gnaiger 2001 Respir Physiol |»Bioblast link«]]
::::» A detailed introduction into high-resolution respirometry with particular emphasis on kinetics and measurements at low oxygen
::::» Mitochondrial kinetics measured by high-resolution respirometry
::::» Calibrations and corrections for response time and instrumental background
::::» Steady-state injection respirometry
::::» Mitochondrial respiratory control at low oxygen
::::» Apparent oxygen affinity and catalytic efficiency of mitochondrial respiration
::::» Effect of ADP and oxygen limitation on ADP/O2 flux ratios
::::» The low-oxygen environment of the cell: Mitochondria between hypoxic and oxidative stress


Gnaiger E, Steinlechner-Maran R, Méndez G, Eberl T, Margreiter R (1995) Control of mitochondrial and cellular respiration by oxygen. J Bioenerg Biomembr 27:583-96. »[[Gnaiger 1995 J Bioenerg Biomembr |Link]]«
:* First publication to establish high-resolution respirometry, based on the OROBOROS Oxygraph.
:* Standardized oxygen calibration (air and internal zero oxygen calibration).
:* Automatic instrumental background correction of oxygen flux.
:* Deconvolution of the oxygen signal for time resolution required in oxygen kinetics.
:* Quality control of the limit of detection of oxygen flux at ±1 pmol O<sub>2</sub>·s<sup>-1</sup>·ml<sup>-1</sup>.


:::* Gnaiger E, Steinlechner-Maran R, Méndez G, Eberl T, Margreiter R (1995) Control of mitochondrial and cellular respiration by oxygen. J Bioenerg Biomembr 27:583-96. - [[Gnaiger 1995 J Bioenerg Biomembr |»Bioblast link«]]
::::» First publication to establish high-resolution respirometry, based on the Oroboros Oxygraph.
::::» Standardized oxygen calibration (air and internal zero oxygen calibration).
::::» Automatic instrumental background correction of oxygen flux.
::::» Deconvolution of the oxygen signal for time resolution required in oxygen kinetics.
::::» Quality control of the limit of detection of oxygen flux at ±1 pmol O<sub>2</sub>·s<sup>-1</sup>·ml<sup>-1</sup>.




Line 109: Line 140:




» [[Media:MiPNet19.18 O2k-Core Manual.pdf|'''O2k-Core Manual.pdf''']]: O2k-Manual for [[DatLab 6]].
:::* [[Media:MiPNet19.18 O2k-Core Manual.pdf|'''O2k-Core Manual.pdf''']]: O2k-Manual for [[DatLab 6]].


» [[MiPNet17.09 IOC Questions | '''Worksheet O2k-Basic''']]: This worksheet contains questions on the O2k and HRR addressed to participants of O2k-Basic Workshops. Answers to these questions are elaborated during the O2k-Workshop.
:::* [[MiPNet17.09 IOC Questions |'''Worksheet O2k-Basic''']]: This worksheet contains questions on the O2k and HRR addressed to participants of O2k-Basic Workshops. Answers to these questions are elaborated during the O2k-Workshop.


» [[O2k-Publications|'''O2k-Publications''']]
:::* [[O2k-Publications: Topics |'''O2k-Publications''']]


» [[O2k-Protocols|'''O2k-Protocols''']]
:::* [[O2k-Protocols |'''O2k-Protocols''']]






[[Category:OroboPedia]]
[[Category:OroboPedia]]

Revision as of 00:23, 31 December 2020

                



IOC recommended reading

COST Action MitoEAGLE

MitoEAGLE 'Mitochondrial physiology' BEC 2020.1

  • Gnaiger Erich et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1. doi:10.26124/bec:2020-0001.v1. - »Bioblast link«


O2k-Workshops.png
O2k-Publications in the MiPMap


Workshop material

  • Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibres from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782 (Palmeira CM, Moreno AJ, eds): Mitochondrial Bioenergetics, 978-1-4939-7830-4. - »Bioblast link«
  • Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. Bioenerg Commun 2020.2. - »Bioblast link« - A new edition with updated terminology and more ..
  • Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. Biomolecules 5:1319-38. »Bioblast pdf«
  • Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81. - »Bioblast link«
  • O2k-Manual.pdf: O2k-Manual for DatLab 7.


HRR: high-resolution respirometry and SUIT protocols

  • Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibres from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782 (Palmeira CM, Moreno AJ, eds): Mitochondrial Bioenergetics, 978-1-4939-7830-4. - »Bioblast link«
  • Lemieux H, Blier PU, Gnaiger E (2017) Remodeling pathway control of mitochondrial respiratory capacity by temperature in mouse heart: electron flow through the Q-junction in permeabilized fibers. Sci Rep 7:2840, DOI:10.1038/s41598-017-02789-8. - »Bioblast link«
» Updated terminology with SUIT protocols.
» NADH-linked or N-pathways (CI-entry into Q); succinate-linked or S-pathway (CII-entry into Q); NS-pathway (convergent CI&II-entry into Q).
  • Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. BEC 2020.2. - »Bioblast link«
» A hard copy of this monograph on mitochondrial physiology will be included in the workshop material. The Blue Book is available on-line free of charge and provides a background on OXPHOS analysis:
» From oxygen concentration to oxygen flux •• Membrane potential: electric and chemical forces •• Coupling and efficiency •• Biochemical thresholds
» Respiratory states
» MitoPathways - the electron transfer-pathway
» Normalization of mitochondrial respiration: coupling control and pathway control.


  • Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley:327-52. - »Bioblast link«
» Methodological introduction, comparison of HRR with traditional oxygraphy.
» Dramatically different: high-resolution respirometry - the Oroboros O2k.
» Calibration of polarographic oxygen sensors and oxygen concentration in respiration media at air saturation.
» From Oxygraph slopes to respiratory flux corrected for background effects.
» Phosphorylation or coupling control protocol (PCP, CCP) with intact cells.
» Titration steps of the CCP.
» Experimental example.
» Flux control ratios from the CCP.
» Living cells, permeabilized cells and tissue, or isolated mitochondria?




OXPHOS ROUTINE ETS LEAK ROX


O2k-FluoRespirometer

O2k-FluoRespirometry

  • Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. Biomolecules 5:1319-38. »Bioblast pdf«
  • Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81. - »Bioblast link«
  • Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61. - »Bioblast link«
  • Eigentler A, Fontana-Ayoub M, Gnaiger E (2013) O2k-Fluorometry: HRR and H2O2 production in mouse cardiac tissue homogenate. Mitochondr Physiol Network 18.05(01):1-6. - »Bioblast link«


TPP/ISE

  • O2k-MultiSensor System with ion selective electrodes (ISE) MiPNet15.03
  • Determination of membrane potential using TPP+ and an ion selective electrode (ISE) MiPNet14.05
  • Rottenberg H (1984) Membrane potential and surface potential in mitochondria: uptake and binding of lipophilic cations. J Membr Biol 81:127-38.


Publications in the MiPMap

Further reading

  • Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41:1837-45. - »Bioblast link«
» Respirometry with permeabilized fibres and isolated mitochondria.
» Convergent NS (CI&II) electron input and OXPHOS capacity.
» Tissue-OXPHOS capacity in human permeabilized muscle fibres and isolated mitochondria.
» Tissue-OXPHOS capacity and functional diversity.


  • Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopsies of human muscle. Methods Mol Biol 810:25-58. - »Bioblast link«
» Introduction to high-resolution respirometry (HRR), SUIT protocols and flux control ratios.


  • Scandurra FM, Gnaiger E (2010) Cell respiration under hypoxia: Facts and artefacts in mitochondrial oxygen kinetics. Adv Exp Med Biol 662:7-25. - »Bioblast link«
» Isolated mitochondria and intact cells.
» Nanomolar resolution of oxygen concentration.
» Effects of cell density and activity.
» Closed versus open systems.
» p50 in vivo.


  • Gnaiger E, Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Steurer W, Margreiter R (2000) Mitochondria in the cold. In: Life in the Cold (Heldmaier G, Klingenspor M, eds) Springer, Heidelberg, Berlin, New York:431-42. - »Bioblast link«
» Isolated mitochondria and permeabilized muscle fibers.
» Optimization of mitochondrial cold storage • Mitochondrial respiration medium, MiR05 • Mitochondrial cold ischemia-reperfusion injury.


  • Renner K, Amberger A, Konwalinka G, Kofler R, Gnaiger E (2003) Changes of mitochondrial respiration, mitochondrial content and cell size after induction of apoptosis in leukemia cells. Biochim Biophys Acta 1642:115-23. - »Bioblast link«
» Intact cells.
» Cytochrome c oxidase.
» Cytochrome c test.
» Respiration per million cells, per citrate synthase, per mg protein, or per cytochrome c oxidase activity.


  • Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128:277-97. - »Bioblast link«
» A detailed introduction into high-resolution respirometry with particular emphasis on kinetics and measurements at low oxygen
» Mitochondrial kinetics measured by high-resolution respirometry
» Calibrations and corrections for response time and instrumental background
» Steady-state injection respirometry
» Mitochondrial respiratory control at low oxygen
» Apparent oxygen affinity and catalytic efficiency of mitochondrial respiration
» Effect of ADP and oxygen limitation on ADP/O2 flux ratios
» The low-oxygen environment of the cell: Mitochondria between hypoxic and oxidative stress


  • Gnaiger E, Steinlechner-Maran R, Méndez G, Eberl T, Margreiter R (1995) Control of mitochondrial and cellular respiration by oxygen. J Bioenerg Biomembr 27:583-96. - »Bioblast link«
» First publication to establish high-resolution respirometry, based on the Oroboros Oxygraph.
» Standardized oxygen calibration (air and internal zero oxygen calibration).
» Automatic instrumental background correction of oxygen flux.
» Deconvolution of the oxygen signal for time resolution required in oxygen kinetics.
» Quality control of the limit of detection of oxygen flux at ±1 pmol O2·s-1·ml-1.


O2k-Manual

General

  • Worksheet O2k-Basic: This worksheet contains questions on the O2k and HRR addressed to participants of O2k-Basic Workshops. Answers to these questions are elaborated during the O2k-Workshop.