Course on High-Resolution Respirometry 2009 OROBOROS
Mitochondrial Physiology Network 10.6: 1-6 (2005-2009)
Last update: 2009-12-04
The OROBOROS Flow Chart for High-Resolution Respirometry
OROBOROS INSTRUMENTS
high-resolution respirometry
Oxygraph-2k Titration-Injection microPump TIP-2k
Integrated Suction System ISS
O2k
O2k Accessory Box
Erich Gnaiger OROBOROS INSTRUMENTS,
high-resolution respirometry Schöpfstr. 18, 6020 Innsbruck, Austria E-mail:
[email protected] www.oroboros.at
The OROBOROS flow chart for high-resolution respirometry provides a guideline for the use of the Oxygraph-2k Manual and the OROBOROS Protocols. Chapter numbers in the boxes indicate the files in various subdirectories on the OROBOROS CD “Course on High-Resolution Respirometry”, in Mitochondrial Pathways and Respiratory Control (OROBOROS MiPNet Publications, Innsbruck 2007), or in www.oroboros.at. Recommended background reading: Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir. Physiol. 128: 277-297. Gnaiger E, Méndez G, Hand SC (2000) High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. Proc. Natl. Acad. Sci. USA 97: 1108011085. Gnaiger E., Steinlechner-Maran R., Méndez G., Eberl T., Margreiter R. (1995) Control of mitochondrial and cellular respiration by oxygen. J. Bioenerg. Biomembr. 27: 583-596. 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.
1.1.A. Oxygraph Overview.
1.1.B.
1.1.C.
1.1.D. Oxygraph Specifications.
The Oxygraph for high-resolution respirometry. MiPNet 6.1.
DatLab 4 On-Line.
DatLab 4: The fourth dimension of high-resolution O2k respirometry. MiPNet 10.1.
[email protected]
Oxygraph-2k.
Paradigm shift and features of highresolution respirometry. MiPNet 9.1. MiPNetP01: 80-86.
Test experiments on specifications of the OROBOROS Oxygraph. MiPNet 6.5.
www.oroboros.at
Flow Chart – High-Resolution Respirometry
2
O2k arrival, unpacking, and assembly O2k.A. O2k-Start.
Oxygraph-2k: Quick start of highresolution respirometry. MiPNet 12.6.
O2k.2. Manual Update.
O2k-Manual: What is new? MiPNet 12.5.
Get prepared for test runs O2k.B. POS Service.
Service of the polarographic oxygen sensor OROBoPOS. MiPNet 8.4.
Did you calibrate the volume of the O2k chambers?
First test runs O2k.C. DatLab4 Guide.
DatLab 4 Guide through the menues. MiPNet 12.7.
DatLab 4 Oxygen and calibration. MiPNet 12.8.
2.4.C.
pX
2.2.A. Cell Respiration.
An experiment with high-resolution respirometry: Phosphorylation control in cell respiration. MiPNet 10.4. MiPNetP01: 51-61.
Did you install DatLab?
O2k.D. DatLab4 O2kCalibration.
Background reading
2.2.B. Cell Respiration.
High-resolution respirometry with leukemia cells: Routine respiration, respiratory control and coupling. MiPNet 8.9. MiPNetP01: 62-73.
2.4.A. Oxygen Solubility. (pH)
Instrumental Background.
Oxygraph-2k: Instrumental background calibration and accuracy of oxygen flux. MiPNet 14.6.
Oxygen solubility in media. MiPNet 6.3.
experimental
Ref. [1,13,25]
DatLabDemo
Prepare media and solutions [12] 2.3.A. Mitomedium MiR06.
Mitochondrial respiration medium MiR06. MiPNet 14.13.
2.3.C.
Chemicals & Media.
Selected media and chemicals for respirometry with mitochondria and permeabilized cells. MiPNet 3.2.
Prepare titration syringes 2.3.B. O2k Titrations.
Oxygraph-2k titrations: Permeabilized cells and biopsies. MiPNet 9.12. MiPNetP01: 79.
OROBOROS INSTRUMENTS
Course on high-resolution respirometry
Flow Chart – High-Resolution Respirometry
3
O2k.C. DatLab4 Guide.
DatLab 4 Guide through the menues. MiPNet 12.7.
Use DatLab 4 [25]
1.1.C. DatLab 4 On-Line Analysis.
O2k.E. DatLab4 O2 Flux Analysis.
DatLab 4: The fourth dimension of high-resolution O2k respirometry. MiPNet 10.1.
DatLab 4 O2 flux analysis: on-line. MiPNet 12.9.
DatLabDemofile: 2005-04-10 EF-01.DLD
Apply the TIP-2k [9,25]
1.4.A. Titration and Injection.
O2k.F. Titration-Injection microPump TIP-2k.
From step titration to ramp injection: Uncoupling by FCCP with TIP. MiPNet 4.5.
Titration-Injection microPump. TIP-2k manual. MiPNet 12.10.
Clean the chamber O2k not used for days and weeks O2k.D. DatLab4 O2kCalibration. DatLab 4 Oxygen and calibration. MiPNet 12.8.
pX
(pH)
Problem with stability of the POS Page 2
O2k.B. POS Service.
Service of the polarographic oxygen sensor OROBoPOS. MiPNet 8.4.
Problem with instrumental background oxygen flux [1,25] O2k not used for months O2k.A. O2k-Start.
Oxygraph-2k: Quick start of highresolution respirometry. MiPNet 12.6.
Measurement of cytochrome c oxidase activity [5,6,14,17,21] 2.2.A. Cell Respiration.
2.4.D. Chemical Background.
An experiment with high-resolution respirometry: Phosphorylation control in cell respiration. MiPNet 10.4. MiPNetP01: 51-61.
Oxygraph assay of cytochrome c oxidase activity: Chemical background correction. MiPNet 6.6.
DatLabDemofile: 2005-04-09 EF-03.DLD
Measurement of oxygen kinetics • in isolated mitochondria [1,5,6,9,13,14] • in cells [1,2,6,15,20,24]
O2k.I. DatLab2 Manual. DatLab 2 Analysis. MiPNet 2.7.
O2k.G. DatLab2 Time Constant.
Ref. [1,13]
O2k.H. DatLab2 Oxygen Kinetics.
2.2.D. NO Oxygraph-2k Workshop UAB.
High time resolution. MiPNet 2.4.
Analysis of oxygen kinetics. MiPNet 2.5.
OROBOROS INSTRUMENTS
NO effect on mitochondrial oxygen kinetics at low oxygen. MiPNet 8.12.
Course on high-resolution respirometry
Flow Chart – High-Resolution Respirometry
Respiratory control and coupling • in isolated mitochondria [1,9,13] • in intact cells [2,3,18,21,22,25,27] 2.2.A2. Cell respiration and Phosphorylation.
An experiment with high-resolution respirometry: Phosphorylation control in cell respiration. MiPNet 10.4. MiPNetP01: 51-61.
4
2.3.A. Mitomedium MiR06.
Mitochondrial respiration medium MiR06. MiPNet 8.5.
2.2.A1. Cell Respiration.
High-resolution respirometry with leukemia cells: Routine respiration, respiratory control and coupling. MiPNet 8.9. MiPNetP01: 62-73.
Respiration and cell membrane permeabilization • in cells [4,8,18,21] • in muscle fibers [12,23] • in liver tissue [16] 2.1.
MitoPathways (A-E)
In: Mitochondrial pathways and respiratory control. MiPNet Publications 2007: 7-50 (MiPNetP01).
2.4.E.
Mitochondria or Permeabilized Cells.
Isolated mitochondria permeabilized tissues and
or cells.
2.4.G. Permeabilized Fiber Preparation.
Preparation of permeabilized muscle fibers for diagnosis of mitochondrial respiratory function. MiPNet 12.21.
Respiration and relation to marker enzymes [16,21,22,23,27] 2.3.D. Complex I. Complex I MiPNet 8.15.
2.3.E.
laboratory
protocol.
Citrate Synthase.
Citrate synthase - laboratory protocol. MiPNet 8.14.
2.3.F.
Lactate Dehydrogenase.
Lactate dehydrogenase - laboratory protocol. MiPNet 8.18.
Respiration and mitochondrial defects [3,7,8,11,12,16,18,21,22,23,24,27] Respiratory measurement of microbial growth [19] Contact to O2k users www.oroboros.at/index.php?mipnet
OROBOROS INSTRUMENTS
Course on high-resolution respirometry
Flow Chart – High-Resolution Respirometry
5
References 1. 2. 3.
4.
5. 6. 7. 8. 9. 10. 11.
12.
13. 14. 15. 16. 17.
Gnaiger E, Steinlechner-Maran R, Méndez G, Eberl T, Margreiter R (1995) Control of mitochondrial and cellular respiration by oxygen. J. Bioenerg. Biomembr. 27: 583-596. Steinlechner-Maran R, Eberl T, Kunc M, Margreiter R, Gnaiger E (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am. J. Physiol. 271: C2053-C2061. Steinlechner-Maran R, Eberl T, Kunc M, Schröcksnadel H, Margreiter R, Gnaiger E (1997) Respiratory defect as an early event in preservation/reoxygenation injury in endothelial cells. Transplantation 63: 136-142. Gnaiger E, Kuznetsov AV, Lassnig B, Fuchs A, Reck M, Renner K, Stadlmann S, Rieger G, Margreiter R (1998) High-resolution respirometry. Optimum permeabilization of the cell membrane by digitonin. In BioThermoKinetics in the Post Genomic Era (Larsson C, Påhlman I-L, Gustafsson L, eds) Chalmers Reproservice, Göteborg: 89-95. Gnaiger E, Lassnig B, Kuznetsov AV, Margreiter R (1998) Mitochondrial respiration in the low oxygen environment of the cell. Effect of ADP on oxygen kinetics. Biochim. Biophys. Acta 1365: 249-254. Gnaiger E, Lassnig B, Kuznetsov AV, Rieger G, Margreiter R (1998) Mitochondrial oxygen affinity, respiratory flux control and excess capacity of cytochrome c oxidase. J. exp. Biol. 201: 1129-1139. Gnaiger E, Rieger G, Stadlmann S, Amberger A, Eberl T, Margreiter R (1999) Mitochondrial defect in endothelial cold ischemia/reperfusion injury. Transplant. Proc. 31: 994-995. Oexle H, Gnaiger E, Weiss G (1999) Iron-dependent changes in cellular energy metabolism: influence on citric acid cycle and oxidative phosphorylation. Biochim. Biophys. Acta 1413: 99-107. Gnaiger E, Méndez G, Hand SC (2000) High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. Proc. Natl. Acad. Sci. USA 97: 11080-11085. Gnaiger E, Kuznetsov AV, Königsrainer A, Margreiter R (2000) Autooxidation of glutathione in organ preservation solutions. Transplant. Proc. 32: 14. Gnaiger E, Kuznetsov AV, Rieger G, Amberger A, Fuchs A, Stadlmann S, Eberl T, Margreiter R (2000) Mitochondrial defects by intracellular calcium overload versus endothelial cold ischemia/reperfusion injury. Transpl. Int. 13: 555-557. 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, Heiderlberg, Berlin, New York, pp. 431-442. Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir. Physiol. 128: 277-297. Gnaiger E, Kuznetsov AV (2002) Mitochondrial respiration at low levels of oxygen and cytochrome c. Biochem. Soc. Trans. 30: 252-258. Hütter E, Renner K, Jansen-Dürr P, Gnaiger E (2002) Biphasic oxygen kinetics of cellular respiration and linear oxygen dependence of antimycin A inhibited oxygen consumption. Molec. Biol. Rep. 29: 83-87. Kuznetsov AV, Strobl D, Ruttmann E, Königsrainer A, Margreiter R, Gnaiger E (2002) Evaluation of mitochondrial respiratory function in small biopsies of liver. Analyt. Biochem. 305: 186-194. Renner K, Kofler R, Gnaiger E (2002) Mitochondrial function in glucocorticoid triggered T-ALL cells with transgenic Bcl-2expression. Molec. Biol. Rep. 29: 97-101.
OROBOROS INSTRUMENTS
Course on high-resolution respirometry
Flow Chart – High-Resolution Respirometry 18.
19. 20. 21.
22.
23.
24. 25.
26. 27.
28. 29. 30.
31.
6
Stadlmann S, Rieger G, Amberger A, Kuznetsov AV, Margreiter R, Gnaiger E (2002) H2O2-mediated oxidative stress versus cold ischemia-reperfusion: mitochondrial respiratory defects in cultured human endothelial cells. Transplantation 74: 1800-1803. Steininger C, Allerberger F, Gnaiger E (2002) Clinical significance of inhibition kinetics in Streptococcus pyogenes in response to penicillin. J. Antimicrob. Chemother. 50: 517-523. Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv. Exp. Med. Biol. 543: 39-56. 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. Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. Biochem. J. 380: 919-928. Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Mark W, Steurer W, Saks V, Usson Y, Margreiter R, Gnaiger E (2004) Mitochondrial defects and heterogeneous cytochrome c release after cardiac cold ischemia and reperfusion. Am. J. Physiol. Heart Circ. Physiol. 286: H1633–H1641. Pecina P, Gnaiger E, Zeman J, Pronicka E, Houštěk J (2004) Decreased affinity to oxygen of cytochrome c oxidase in Leigh syndrome caused by SURF1 mutations. Am. J. Physiol. Cell Physiol. 287: C1384-C1388. Garedew A, Hütter E, Haffner B, Gradl P, Gradl L, Jansen-Dürr P, Gnaiger E (2005) High-resolution respirometry for the study of mitochondrial function in health and disease. The OROBOROS Oxygraph-2k. Proc. 11th Congress Eur. Shock Soc. Vienna, Austria (Redl H, ed), Medimond Internat. Proc., Bologna: 107-111. Hütter E, Unterluggauer H, Garedew A, Jansen-Dürr P, Gnaiger E (2006) Highresolution respirometry - a modern tool in aging research. Exp. Gerontol. 41: 103-109. Stadlmann S, Renner K, Pollheimer J, Moser PL, Zeimet AG, Offner FA, Gnaiger E (2006) Preserved coupling of oxydative phosphorylation but decreased mitochondrial respiratory capacity in IL-1β treated human peritoneal mesothelial cells. Cell. Biochem. Biophys. 44: 179-186. Boushel R, Gnaiger E, Schjerling P, Skovbro M, Kraunsøe R, Dela F (2007) Patients with Type 2 Diabetes have normal mitochondrial function in skeletal muscle. Diabetologia 50: 790-796. Gnaiger E, ed (2007) Mitochondrial Pathways and Respiratory Control. OROBOROS MiPNet Publications, Innsbruck: 96 pp. Electronic 1st ed ISBN 9783-9502399-0-4 Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and highresolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley: 327-352. Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int. J. Biochem. Cell Biol. 41: 1837–1845.
OROBOROS INSTRUMENTS
Course on high-resolution respirometry
Flow Chart – High-Resolution Respirometry
OROBOROS INSTRUMENTS
7
Course on high-resolution respirometry
