IOC recommended reading

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IOC recommended reading

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HRR: high-resolution respirometry

Recommended reading

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. - »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 to electron transfer complexes
  • Normalization of mitochondrial respiration: coupling control and substrate 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 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?


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



O2k-Publications in the MiPMap

SUIT protocols for high-resolution respirometry

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. - »Bioblast link«

  • The Blue Book: OXPHOS analysis; CI-linked, CII-linked, CI&II-linked pathways, respiratory states, abbreviations.


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. - »Bioblast link«

  • Introduction to high-resolution respirometry (HRR), SUIT protocols and flux control ratios.


» MitoPedia: Respiratory states OXPHOS ROUTINE ETS LEAK ROX


O2k-Fluorometer

O2k-Fluorometry

  • 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: 1837-1845. - »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.


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, 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

» O2k-Core Manual.pdf: O2k-Manual for DatLab 6.
» 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-Protocols
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