IOC recommended reading

General

»O2k-Core Manual.pdf

»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

»Respiratory states

SUIT protocols for high-resolution respirometry

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

• 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: 327-352. »Open Access
  • Methodological introduction to HRR, with focus on Polarographic oxygen sensor and traditional oxygraphy
  • Dramatically different: High-resolution respirometry - the 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 (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?

HRR and O2k-Fluorometry

• »Manual: O2k-Fluorescence LED2-Module
• Krumschnabel 2014 Methods Enzymol
• 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. »Open Access
• »O2k-Fluorometry Publications

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

• Calculation of mitochondrial membrane potential from measurements with a TPP electrode

• Rottenberg H (1984) Membrane potential and surface potential in mitochondria: uptake and binding of lipophilic cations. J Membr Biol 81: 127-138.

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. »Abstract
  • Respirometry with permeabilized fibres and isolated mitochondria
  • Convergent CI+II electron input and OXPHOS capacity
  • Tissue-OXPHOS capacity in human permeabilized muscle fibres and isolated mitochondria
  • Tissue-OXPHOS capacity and functional diversity

• Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128: 277-297. – A detailed introduction into high-resolution respirometry with particular emphasis on kinetics and measurements at low oxygen. »Abstract
  • 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

• Scandurra FM, Gnaiger E (2010) Cell respiration under hypoxia: Facts and artefacts in mitochondrial oxygen kinetics. Adv Exp Med Biol 662: 7-25. »Open Access
  • 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: 431-442. »Abstract
  • Isolated mitochondria and permeabilized muscle fibers, MiR05
  • 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: 115-123. »Abstract
  • Intact cells
  • Cytochrome c oxidase
  • Cytochrome c test
  • Respiration per million cells, per citrate synthase, per mg protein, or per cytochrome c oxidase activity.