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
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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.
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24.
25.
26.
27.
28.
29.
30.
31.
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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