IOC recommended reading

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. »Open Access«

• A hard copy of this monography 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 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?

Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128:277-97. »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

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. »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. »Bioblast Access«

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

» MitoPedia: Respiratory states

O2k-Fluorometry

• »Manual: O2k-Fluorometry
• Krumschnabel et al (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. »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. »Open Access«
• 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-Publications: O2k-Fluorometry

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 81:127-38.

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. »Link«

• 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.

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:431-42. »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. »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. »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.

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