O2k-Protocols
Mitochondrial Physiology Network 10.04(08):1-12 (2015)
Updates: www.bioblast.at/index.php/MiPNet10.04_CellRespiration
2005-2015 OROBOROS
Version 8: 2015-01-11
An experiment with
high-resolution
respirometry:
coupling control in
cell respiration
O2k-Workshop Report, IOC30, Schroecken, Austria.
Gnaiger E
1
OROBOROS INSTRUMENTS Corp, high-resolution
respirometry
Schöpfstr 18, A-6020 Innsbruck, Austria
erich.gnaiger@oroboros.at; www.oroboros.at
2
Medical University of Innsbruck
D. Swarovski Research Laboratory
6020 Innsbruck, Austria
Section
1.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Introduction ............................................... 1
The coupling control protocol ....................... 2
The cells .................................................... 2
Air calibration ............................................. 3
Fill the O2k-Chambers ................................. 3
DatLab recording ........................................ 4
Manual titration .......................................... 4
Automatic step-titrations with the TIP2k ....... 5
DatLab real-time analysis ............................ 7
Instrumental background ............................. 8
Chemical background................................... 8
Results and discussion ................................. 9
Selected references ................................... 12
Page
Introduction
Methodological and conceptual features of highresolution respirometry are illustrated in an experiment
with cultured, suspended cells in the OROBOROS
Oxygraph-2k (O2k). The experiment demonstrates
manual titrations of inhibitors and automatic uncoupler
titrations using the Titration-Injection microPump
TIP2k. Application of the DatLab software is shown for
instrumental control (O2k and TIP2k) and real-time
data analysis. The following guideline describes the
experiment in the form of a laboratory protocol,
instruments@oroboros.at
www.oroboros.at
MiPNet10.04
Cell respiration and coupling control
2
complementary to the O2k-Manual. The experiments
were carried out by participants of an O2k-Workshop
on high-resolution respirometry (IOC30; Schröcken,
Austria; April 2005).
2.
3.
The coupling control protocol
The cells
A simple coupling control protocol (CCP) is applied for
evaluation of the ROUTINE physiological control state of
intact cell respiration (R), LEAK respiration (L),
noncoupled respiratory capacity through the electron
transfer system, ETS (E), and rotenone+antimycin Asensitive respiration to correct mt-respiration by
residual oxygen consumption (ROX). When using cells
suspended in culture medium, respiration is supported
by respiratory substrate in the medium, whereas in a
crystalloid
medium
without
energy
substrate
(mitochondrial respiration medium, MiR06; Gnaiger et
al 2000) cells respire on endogenous substrates. In the
latter case, the effect of an intracellular ion composition
on cell respiration must be evaluated (no difference
between culture medium and mitochondrial medium is
observed for R of endothelial cells; Stadlmann et al
2002), and the respiratory protocol can be extended to
obtain a measure of enzyme activity of cytochrome c
oxidase (Renner et al 2003). Application of cell culture
medium for respiratory measurements is advantageous
when aiming at near-physiological conditions of intact
cells.
The basic PC-protocol takes about 90 min,
including (1) a 10-min period of ROUTINE respiration,
reflecting the aerobic metabolic activity under cellular
routine conditions (state R), (2) the oligomycininhibited LEAK respiration, which is caused mainly by
compensation for the proton leak after inhibition of ATP
synthase (state L); (3) uncoupler titration with the
TIP2k, which yields the maximum stimulated
respiration as a measure of ETS capacity of noncoupled
mitochondria in non-permeabilized cells (state E), and
quantitatively describes the dependence of respiration
on uncoupler concentration; (4) rotenone- and (5)
antimycin A-inhibited respiration after sequential
inhibition of Complex I and III, as an estimate of
residual oxidative side reactions (ROX).
Inhibitors and the uncoupler applied in this
protocol are freely permeable through the intact plasma
membrane and do not require, therefore, cell
membrane permeabilization (Hütter et al 2004).
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Cell respiration and coupling control
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Parental hematopoietic 32D cells and v-Raf transformed
32D (32D-v-Raf) cells were used in dilute suspension.
32D is an immortalized mouse promyeloid cell line
originally derived from long-term cultures of murine
bone marrow, grown in RPMI supplemented by WEHI3B conditioned medium as a source of IL-3 (Greenberg
et al 1983). These cells have a requirement for IL-3 to
remain undifferentiated. Removal of IL-3 leads to cell
cycle arrest in the G0/G1 phase, followed by induction of
apoptosis (Troppmair and Rapp 2003).
4.
Air calibration
Set up the Oxygraph-2k at 37 °C (MiPNet19.01A O2kStart), clean and wash the O2k chambers after
overnight storage in 70% ethanol. Air calibration of the
polarographic oxygen sensors (OroboPOS, POS) is
performed in culture medium RPMI (MiPNet06.03,
MiPNet19.01D). The Integrated Suction System (ISS) is
applied to siphon off medium from the chambers during
the step-wise washing procedure. The level of the
medium in the wash bottle must not be allowed to
increase above the mark given by the stainless steel
housing of the ISS. Aqueous medium must not be
drawn into the filter of the ISS. Otherwise the suction
power is reduced to zero, and the filter must be dried
by applying air pressure and a gas flow through the
disconnected filter.
After air calibration close the
O2k-Chambers and record oxygen consumption by the
POS at air saturation over 15 min. The slope
(unocrrected) should stabilize at 2 to 3 pmol∙s-1∙ml-1,
which
corresponds
to
the
theoretical
oxygen
consumption by the POS and provides a quality control
for the medium and appropriately sterile state of the
O2k-Chambers. A more general quality control for O2k
performance is provided in an instrumental background
test, usually carried out the day before (Gnaiger 2001).
In the O2k-Workshop, instrumental and chemical
background tests are carried out on day 2
(MiPNet14.06).
After completion of air calibration, disconnect
DatLab [F7] to interrupt data recording, and close the
file with the calibration information.
5.
Fill the O2k-Chambers
A suspension of cells in culture medium (RPMI) is added
into the Oxygraph-2k chambers at a concentration such
that ROUTINE respiration yields a volume-specific
OROBOROS INSTRUMENTS
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Cell respiration and coupling control
4
oxygen flux of about 20 pmol∙s-1∙cm-3 (0.5106 cells ml-1
with 32D cells).
Siphon off the medium from the chambers and add
3 ml cell suspension (32D or 32D-v-Raf in RPMI) into
each chamber, while rotation of the stirrers is
maintained in the Oxygraph-2k. Samples can now be
collected from the O2k-chambers containing a
homogenous cell suspension, for analysis of cell count,
protein concentration, and enzyme assays (e.g.
Complex I, (MiPNet08.15) CS (MiPNet08.14) and LDH
(MiPNet08.18). The volume of cell suspension
remaining in the chamber must be at least 2.1 ml.
Close the chambers by fully inserting the stoppers
into the volume-calibrated position (gentle twisting of
the stoppers clockwise and anticlockwise), thereby
extruding all gas bubbles. Siphon off any excess cell
suspension from the receptacle of the stoppers. As a
recommendation
for
avoiding
any
external
contamination, place the Perspex covers on top of the
stoppers. This precaution is, however, without any
consequence on oxygen diffusion into the chamber.
6.
DatLab recording
In DatLab, connect the O2k for data recording (press
the function key [F7] to open the “O2k-MultiSensor
Control” window). Use the sequential number for the
DatLab file name. Edit the “Experiment” window
(MiPNet19.01E). For the background parameters,
standard values may be used initially (intercept a° = 2.0; slope b° = 0.025). In particular, enter the cell
density, as a basis for real-time display of respiratory
activity per million cells, corrected for instrumental
background. For this purpose, select the graph layout
“Specific flux per unit sample”. (Since a cell counter
may not be available at the O2k-Workshop, we use an
approximate estimate of cell density).
Over an initial period of 20 min, the respiratory
flux stabilizes and attains a constant level of ROUTINE
respiration (Fig. 1). Set a mark on the plot for oxygen
flow over a period of constant respiration. Rename the
mark as “1-R” (MiPNet19.01E). This first mark on the
plot defines the section of the experiment in respiratory
state R. The Marks\Names function are used to edit
mark names from a template (CCP with TIP2k 22A2).
7.
Manual titration
Titrate of 1 µl oligomycin (Omy) into each chamber
with a 10 µl Hamilton syringe (4 mg∙ml-1 stock in EtOH,
OROBOROS INSTRUMENTS
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Cell respiration and coupling control
5
2 µg∙ml-1 final Omy concentration; MiPNet09.12). For
titrations, the needle with standard length must be fully
inserted through the capillary of the stopper. Set an
event at the time of Omy titration [F4]. After inhibition
of ATP synthase, flux declines to a new steady state,
although flux in state L tends to increase gradually with
time. Set a second mark on the stable oxygen flux
(mark name “2-L”; Fig. 1).
8.
Automatic step-titrations with the TIP2k
Titration of uncouplers must be performed carefully,
since optimum uncoupler concentrations have to be
applied to achieve maximum stimulation of flux,
avoiding over-titration which results in inhibition of
respiration. Optimum uncoupler concentrations depend
on the cell type, cell concentration, medium, and are
different in permeabilized versus unpermeabilized cells.
Highest accuracy is achieved by step-titrations of small
volumes of uncoupler, and intermittent observation of
the effect on instantaneous respiration. The titration is
terminated when a small increase of uncoupler
concentration does not yield a further stimulation of
oxygen flux (Steinlechner-Maran et al 1996). The
OROBOROS
Titration-Injection
microPump
TIP2k
provides an accurate and convenient tool for automatic
performance of such step-titrations (Fig. 1).
8.1. Operation of the TIP2k
Two Hamilton syringes with 27 mm needle length and
0.09 mm needle inner diameter are mounted on the
TIP2k for simultaneous titrations into the two O2k
chambers. The TIP2k syringes are filled with an
uncoupler stock solution (10 mM FCCP in pure ethanol;
ten-fold higher concentration than recommended for
manual titrations) up to the 100 µl mark
(MiPNet12.10).
8.2. TIP2k setup in DatLab
In DatLab, the “TIP Control” window [F8] is edited to
obtain the following setup configuration (MiPNet12.10):
Vol.[µl]
0.1 (this is the volume added during each titration cycle
and corresponds to an increase of the final
concentration of 0.5 µM FCCP in the O2k-Chamber).
Flow [µl/s] 20 µl/s.
Delay [s]
0 (this is the time between the start of the titration
program and the first titration; to minimize washout
effects it is preferable to start without delay).
Interval [s] 120 (this is the time interval of a single titration cycle).
OROBOROS INSTRUMENTS
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Cell respiration and coupling control
6
Cycles
15 (this is the number of repetitions of a titration cycle;
15 titration steps correspond to a final concentration of
7.5 µM).
Solvent
Ethanol
Substance FCCP (updated recommendation: CCCP)
Conc. in TIP 10 mM
Edit the TIP setup name and save the setup
information.
8.3. TIP2k titration
Before inserting the TIP2k needles into the O2kChambers, press [F8] in DatLab to open the TIP-Control
window, click on “Test start”, clean the needle tips with
absolute ethanol, and insert the needles partially (c. 3
cm) into the stopper capillary for about 2 min. Then
insert the needles to the positioning ring: The needle
tips protrude into the chamber without touching the
stirrer. Start the TIP2k program by pressing “start” in
the F8-window. Automatic events are set in the graphs
by DatLab for each titration step.
Mark the stable section of oxygen flux after each
titration step, and edit the name of the mark according
to the total uncoupler concentration that has already
been titrated up to this step. For example, “U0.5”;
“U1.0”; “U1.5” etc. The marks can be renamed
automatically from a template (Marks\Names; template
“CCP with TIP2k 22A2”).
Parallel experiments were run in the two O2kChambers at identical cell densities. In Fig. 1, the JO2
traces (A) and oxygen concentration [O2] (B) from the
two chambers are superimposed for illustrating the
precision of high-resolution respirometry.
Oxygen concentration during the titration must not
decline close to zero levels. Else the titration must be
interrupted for re-oxygenation, before continuation of
the titration protocol. When a plateau of flux is reached
and further titrations of uncoupler do not stimulate or
even inhibit respiration, press [F8] and stop the TIP2k
titration. Mark the section of maximum flux as 3-E (ETS
capacity). Avoid excess uncoupler beyond the optimum
concentration for maximum flux (unless inhibition by
uncoupler of respiration is to be demonstrated).
At the end of the titration, remove the needles
carefully from the chamber. After an aerobic-anoxic
transition (zero oxygen calibration, with a mark R0 on
the plots for oxygen concentration), lift the stoppers
(using the stopper spacer) for re-oxygenation, close the
chamber when the desired oxygen level has been
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MiPNet10.04
Cell respiration and coupling control
7
retained, allow for a 10-min stabilization period, and
continue with manual titrations of rotenone (1 µl of a
0.2 mM Rot stock in ethanol for full inhibition of
Complex I) and antimycin A (1 µl of a 5 mM Ama stock
in ethanol). Mark the sections of inhibited flux (“4-ROX”
after immediate addition of both inhibitors; Fig. 1).
9.
DatLab real-time analysis
100
A
80
80
60
60
40
40
20
20
0
0
0:15
0:30
Omy
Omy
Routine
200
Leak
0:45
1:00
Range [h:min]: 1:30
1:15
needle
P01inP02P03 P04P05P06 P07P08P09 P10P11P12 P13P14P15 P16P17P18
TIP
P19
Stop
F0.5F1.0F1.5F2.0F2.5F3.0F3.5F4.0F4.5F5.0F5.5F6.0
ETS F6.5F7.0F7.5F8.0F8.5F9.0
reox
1:30
RotAma
nmt
200
B
160
160
120
120
80
80
40
40
0
O2 Concentration (F) [nmol/ml]
100
O2 Flow per cells (F) [pmol/(s*Mill)]
Real-time analysis is achieved and a graphical summary
of the results is obtained by exporting the respiratory
flux in the marked sections of the experiments into the
DatLab-Excel template “O2k-Analysis_Cells_0809.xls”
(MiPNet08.09). Open this spread sheet from the
subdirectory
\2.O2kProtocols\Files_Protocols\MiPNet10.04\.., save the xls
file under the subdirectory generated for the
0
0:15
0:30
Omy
Omy
0:45
1:00
Range [h:min]: 1:30
1:15
needle
P01inP02P03 P04P05P06 P07P08P09 P10P11P12 P13P14P15 P16P17P18
TIP
P19
Stop
reox
1:30
RotAma
R0
6
Figure 1. Respiration of 32D cells (1.110 ml-1). Traces
for two chambers of the Oxygraph-2k are superimposed.
A:
Oxygen
flow
[pmol O2s-1106];
B:
Oxygen
concentration [µM] (graph layout “7 Gr1-Flux Gr2-O2
Conc”). After inhibition of ATP synthase with oligomycin,
uncoupler was titrated at 0.5 µM steps with the TIP2k.
Each titration step is automatically marked by an event
(vertical lines). After the aerobic-anoxic transition, O2kChambers were opened for re-oxygenation, causing a 10min disturbance of the traces of respiratory flow. The
TIP2k needles were removed before opening, and reinserted after closing the chambers (see Gnaiger 2008).
OROBOROS INSTRUMENTS
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MiPNet10.04
Cell respiration and coupling control
8
experimental files (MiPNet12.10).
Select the DatLab graph layout “6-Specific flux per
unit sample”, to plot oxygen concentration and flux on
a graph for each separate chamber. Numerical analysis
and a graphical representation of the experiment are
complete at the time of terminating the experiment.
10. Instrumental background
An instrumental background experiment is performed
on the second day of the O2k-Workshop, using
mitochondrial respiration medium MiR06 (MiPNet14.13)
without biological sample, and starting with the
standard protocol for calibration of the oxygen sensor.
Subsequent to testing for POS sensor performance,
the instrumental background test yields a calibration of
the O2k chamber performance.
11. Chemical background
After completing the instrumental background test, the
chambers are partially opened (again using the O2kspacer) for re-oxygenation to near air saturation (not
for re-calibration purposes). After closing the chambers
by lowering the stoppers, flux is allowed to stabilize (10
min). Cytochrome c, ascorbate, and TMPD are manually
injected with Hamilton syringes through the stopper
capillary, to measure chemical autooxidation with the
substrates used for determination of the activity of
cytochrome c oxidase. Autooxidation is strongly
oxygen-dependent, and the reaction is allowed to
proceed over the lunch break.
Chemical background oxygen flux is a linear
function of oxygen concentration above 40-50 µM
(MiPNet06.06). Subsequent to an initial overshoot of
flux as observed occasionally, marks are set at regular
intervals (it is recommended to select the plot for
oxygen concentration for adding these marks, T01,
T02, ..; Fig. 2), only until the critical oxygen
concentration of c. 40-50 µM is reached. If the
experiment proceeds to anoxia, a final mark “R0” is set
for zero calibration (Fig. 2).
Results are displayed in DatLab and the Excel
template “O2k-Background.xls” (use the table
“Template
Chem+O2k-Backgr.”),
for
total
instrumental + chemical background effects (Fig. 2).
The combined parameters, a°’=a°+a’ and b°’=b°+b’
(Fig. 2), are used for real-time instrumental and
chemical background correction in COX activity
determinations, whereas results of the complex
OROBOROS INSTRUMENTS
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MiPNet10.04
Cell respiration and coupling control
9
chemical reaction of autooxidation are obtained after
correction for instrumental background.
12. Results and discussion
The linear parameters a’ and b’ (chemical background,
after correction for instrumental background) are
characteristic for the chemical process in the particular
medium. The mean ±SD from six Oxygraph-2k
chambers with MiR05 (three instruments operated in
parallel by participants of the O2k course) were: a’ =
10.7±1.4 and b’ = 0.24±0.07.
Cellular respiration (oxygen flow) and respiratory
flux control ratios (mean ± SD) are shown for each cell
type (from six Oxygraph-2k chambers each; Fig. 3).
The high reproducibility is remarkable (compare Fig.
1A). Residual oxygen consumption after inhibition of
uncoupled respiration with rotenone and antimycin A
(ROX) was 3% of noncoupled cellular respiration (E’;
Fig. 3A). The relative contribution of ROX was
considerable, however, when related to respiration
87,60
0,2373
200
50
160
40
120
30
80
20
40
10
0
Background O2 flux [pmol.s-1.cm-3]
22,94
10,41
02:40:00
03:20:00
Range [hh:mm:ss]: 4:00:00
04:00:00
04:40:00
y = 0,2394x + 10,229
40
30
20
10
0
0
02:00:00
Close
05:20:00
0
Ascorbat
Cytc TMPD
T01 T02 T03 T04 T05 T06 T07 T08 T09 T10
R0
Comments:
100
150
200
22,46
0,21
87,60
0,2120
50
160
40
120
30
80
20
40
10
0
0
02:46:40
03:28:20
Range [hh:mm:ss]: 4:10:00
04:10:00
04:51:40
05:33:20
Cytc
Ascorbat
TMPD
T01 T02 T03 T04 T05 T06 T07 T08
T09 T10
R0
Comments:
Background O2 flux [pmol.s-1.cm-3]
y = 0,2783x + 3,4406
200
02:05:00
Mark
01
2005-04-10 EF-01.DLD
Averages Unit
R0
Start
05:25:05
Stop
05:33:15
N Points
245
O2 Concentration
nmol/ml
(E)
-0,11
O2 Slope uncorr.
pmol/(s*ml)
(E)
0,02
X O2 Concentration
nmol/ml
(F) -0,05
O2 Slope uncorr.
pmol/(s*ml)
(F)
0,22
Block Temp.°C
37,00
Barom. Pressure
kPa
87,44
Peltier Power%
-25,89
pX (E)
V
-9,99
Slope X (E) Unit
0
pX (F)
V
-9,99
Slope X (F) Unit
0
50
y = 0,2783x + 3,4406
O2 Slope uncorr. (F) [pmol/(s*ml)]
O2 Concentration (F) [nmol/ml]
50
O2 concentration, c O2 [µM]
Chemical Background
Date
Chamber
F
Run
1
2005-04-10 EF-01.DLD
Calib. D
7,7646
0,0122
Backgr. D
-1,63
0,0270
Paste DatLab graph here, reduce to width 15 cm
Close
Mark
01
02
2005-04-10 EF-01.DLD
Averages Unit
R0
R1
Start
04:54:12 00:25:02
Stop
05:05:48 00:27:06
N Points
349
62
X O2 Concentration
nmol/ml
(E) -0,04 165,50
O2 Slope uncorr.
pmol/(s*ml)
(E)
0,07
0,03
O2 Concentration
nmol/ml
(F)
0,86 165,50
O2 Slope uncorr.
pmol/(s*ml)
(F)
1,01
-0,02
Block Temp.°C
37,00
37,00
Barom. Pressure
kPa
87,46
87,6
Peltier Power%
-28,12
-37,93
pX (E)
V
-9,99
-9,99
Slope X (E) Unit
0,00
0,00
pX (F)
V
-9,99
-9,99
Slope X (F) Unit
0,00
0,00
50
y = 0,2394x + 10,229
O2 Slope uncorr. (E) [pmol/(s*ml)]
O2 Concentration (E) [nmol/ml]
Chemical Background
Date
Chamber
E
Run
1
2005-04-10 EF-01.DLD
Calib. C
7,6092
0,0192
Backgr. C
-1,28
0,0232
Paste DatLab graph here, reduce to width 15 cm
40
30
20
10
0
0
50
100
150
02
R1
00:25:24
00:27:37
67
165,514
-0,0881
165,503
0,031
37,0001
87,6
-37,96
-9,99
0
-9,99
0
200
O2 concentration, c O2 [µM]
Fig. 4. Chemical background in MIR05 with cytochrome c, ascorbate and TMPD in the
Figure
Chemical
in MiR05 with
Oxygraph-2k with 2 ml
chamber2.
volume,
using the background
file “O2k-Background.xls”.
cytochrome c, ascorbate and TMPD in the Oxygraph-2k
with 2 ml chamber volume, using the table sheet
“Template Chem+O2k-Backgr.” in the file “O2kBackground.xls”.
inhibited by oligomycin: ROX/L’ = 0.13±0.01 and
0.11±0.01 in the two cell types. Considering the
oxygen dependence of residual respiration (Gnaiger
2003), the oxygen level should be carefully chosen for
OROBOROS INSTRUMENTS
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Cell respiration and coupling control
I O2 [pmol.s -1.10 -6 cells]
100
80
60
40
20
0
Routine
Leak
ETS
ROX
nmt
Respiratory States
Flux Control Ratio
0.4
0.3
0.2
0.1
0.0
L/E
Respiratory States
Figure 3. Respiration of 32D and 32Dv-Raf cells at an experimental cell
density of 1.1106 cells ml-1 (mean 
SD, N=6 for each cell type).
Respiration
of
intact
cells
was
measured under routine conditions (R,
ROUTINE), inhibition by oligomycin (L,
LEAK), uncoupling to maximum flux
(E, electron transfer system), and
inhibition by rotenone and antimycin A
(ROX).
A. Cellular oxygen flow, IO2 [pmols1
10-6 cells], ROX-corrected.
0.5
R/E
10
netR/E
B. Flux control ratios, FCR, ROXcorrected: R/E = IR/IE
L/E = IL/IE
netR/E = (R-L)/E
The inversed FCR (ROX-corrected)
were:
32D
32D-v-Raf
UCR (E/R)
2.60.1
2.70.1
RCR (E/L) 10.01.6
12.23.9
these measurements, particularly avoiding high oxygen
concentrations (Fig. 1).
Mitochondria contribute to residual oxygen
consumption (particularly related to ROS production)
after inhibition of Complexes I and III, which argues
against correcting respiration in states R, L and E for
ROX observed after addition of inhibitors (Stadlmann et
al 2002; 2006; Renner et al 2003; Hütter et al 2004).
Uncoupling prior to inhibition by rotenone and
antimycin A, however, prevents the large increase in
mitochondrial ROS production known to occur in the
presence of rotenone and particularly antimycin A in
isolated coupled mitochondria (Boveris and Chance
1973; Garait et al 2005). Our more recent findings on a
comparison of respiration with intact and permeabilized
cells showed that residual respiration was significantly
lower in permeabilized cells. This suggests that a large
contribution to ROX is not due to mitochondria (which
remain intact after cell membrane permeabilization),
but is related to non-mitochondrial, cellular oxygen
consuming processes (Gnaiger 2003). Consequently,
flux control ratios (FCR) were corrected for ROX (Fig.
3).
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Cell respiration and coupling control
11
The ROUTINE FCR, R/E, of about 0.4 (Fig. 3B)
indicates that 40% of electron transfer system capacity
is utilized in the ROUTINE respiratory state of the intact
cells, whereas the LEAK FCR, L/E, of about 0.1 (Fig. 3B)
indicates that 10% of electron transfer system capacity
is related to non-phosphorylating respiration (mainly
due to proton leak). The net ROUTINE FCR, netR/E,
shows that 30% of total (noncoupled) ETS capacity is
functionally related to the control of respiration by
phosphorylation. By comparison, the netR/E is 0.2 in
fibroblasts (corrected for ROX; Pesta and Gnaiger
2012).
13. Selected references
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. »
Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and
phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol
128:277-97.
Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of
mitochondrial physiology. Adv Exp Med Biol 543:39-55. »
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. »
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. »
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.
»
Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescenceassociated changes in respiration and oxidative phosphorylation in primary human
fibroblasts. Biochem J 380:919-28. »
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. »
Renner K, Amberger A, Konwalinka G, 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. »
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 74:1800-3.
»
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. »
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:C2053-61. »
OROBOROS INSTRUMENTS
O2k-Protocols
MiPNet10.04
»
»
»
»
»
»
»
»
»
»
»
»
»
O2k-Manual
MiPNet19.18A
MiPNet19.18D
MiPNet19.18E
MiPNet12.10
Cell respiration and coupling control
12
Oxygraph-2k: start high-resolution respirometry.
Oxygen calibration by DatLab.
Oxygen flux analysis: real-time.
Titration-Injection microPump TIP2k.
O2k-Protocols
MiPNet06.03 POS: calibration, accuracy and quality control SOP.
MiPNet08.09 Cell respiration.
MiPNet14.06 Instrumental Background.
MiPNet06.06 Chemical Background.
MiPNet08.14 Citrate synthase – laboratory protocol.
MiPNet08.15 Complex I – laboratory protocol.
MiPNet08.18 Lactate dehydrogenase – laboratory protocol.
MiPNet09.12 O2k-Titrations.
MiPNet14.13 Mitochondrial respiration medium – MiR06.
Acknowledgements:
I thank Brigitte Haffner and Eveline Hütter for technical assistance,
Jakob Troppmair for placing the cell cultures at our disposal and all
IOC30 participants for their cooperation.
More details
on the IOC30 experiment » Gnaiger 2008
Full version: go Bioblast
» www.bioblast.at/index.php/MiPNet10.04_CellRespiration
OROBOROS INSTRUMENTS
O2k-Protocols
MiPNet10.04
Cell respiration and coupling control
13
Supplement A. O2k-DatLab Analysis template
IOC-30
Sample
Condition
O2 Concentration (E) [nmol/ml]
120
100
Conc. or Amount
Info
Unit
1,1
25,202
9,677
2005-04-09 EF-03.DLD
Averages
100
80
87,6
0,2443
200
100
160
80
120
60
80
40
40
20
OO22flux
flux
Sample
O2 Flow per cells (E) [pmol/(s*Mill)]
Chamber
Run
#
type
2005-04-09 EF-03.DLD
Info left chamber
3
32D
intact cells
6,589
0,022
Calib. E
-1,9603
0,0234
Backgr. E
Paste DatLab graph here, reduce to width 15 cm
62,6
80
60
60
40
40
40
59,7
00:28:20
Oligo
01-Rout
02-4o
00:56:40
Range [hh:mm:ss]: 1:25:00
needle
P01in P02 P03 P04 P05 P06 P07 P08 P09 P10 P11 P12 P13 P14 P15 P16 P17 P18 TIP
P19
Stop
F0,5 F1,0 F1,5 F2,0 F2,5 F3,0 F3,5 F4,0 F4,5 F5,0 F5,5 F6,0 F6,5 F7,0 F7,5 F8,0 F8,5 F9,0
Comments:
00:42:30
UCR'
RCR'
C'r/C'ru
01:10:50
X
37,3
28,9
19,4
7,9
3,1
01:25:00
reox.
57,3 55,9
25,8
20
20
20
0
00:14:10
60,3 58,8
55,8
44,4
1,0
C
C r
rO
C Cru
ru
R
A
0,
5
1,
0
1,
5
2,
0
2,
5
3,
0
3,
5
14,
0
4,
5
5,
0
5,
5
6,
0
6,
5
7,
0
7,
5
8,
0
8,
5
9,
0
0
62,4 62,6 62,3 61,5 61,0
60,8 61,9
51,5
60
0
0
Unit
Start
Stop
N Points
(E)
O2 Flow per cells (E)
O2 Concentration (F)
O2 Flow per cells (F)
Block Temp.
Barom. Pressure
Peltier Power
pX (E)
Slope X (E)
pX (F)
Slope X (F)
01-Cr
nmol/ml
pmol/(s*Mill)
nmol/ml
pmol/(s*Mill)
°C
kPa
%
V
Unit
V
Unit
00:16:01
00:18:38
78
130,09
25,80
133,78
24,99
37,00
86,77
-39,93
-9,99
0
-9,99
0
AM
04-AntA
C'rO/C'ru
RCR'p
0,30
2,62
12,38
0,38
0,08
Sample
#
Sample
type
Condition
Conc. or Amount
Info
Unit
100
1,1
100
120
80
IOC-30
Cr
Mark
Info right chamber
3
32D
intact cells
6,821
0,0216
Calib. F
-2,7653
0,0295
Backgr. F
Paste DatLab graph here, reduce to width 15 cm
24,34
3,6949
200
87,4
0,2722
100
160
80
120
60
80
40
40
20
#BEZUG!
100
80
O2 flux
O2 flux
Chamber
Run
2005-04-09 EF-03.DLD
O2 Flow per cells (F) [pmol/(s*Mill)]
Date
61,6
58,6
60
80
60
58,2 56,9
55,6
51,2
43,8
60
40
40
40
20
20
20
61,9 61,3 60,4
60,3 61,0 61,5 61,6
59,8
55,4
2005-04-09 EF-03.DLD
Averages
X
37,9
28,1
25,0
18,5
7,4
2,4
0
0
00:14:10
00:28:20
Oligo
01-Rout
Comments:
02-4o
00:42:30
00:56:40
Range [hh:mm:ss]: 1:25:00
needle
P01in P02 P03 P04 P05 P06 P07 P08 P09 P10 P11 P12 P13 P14 P15 P16 P17 P18 TIP
P19
Stop
F0,5 F1,0 F1,5 F2,0 F2,5 F3,0 F3,5 F4,0 F4,5 F5,0 F5,5 F6,0 F6,5 F7,0 F7,5 F8,0 F8,5 F9,0
01:10:50
01:25:00
reox.
AM
04-AntA
UCR'
RCR'
C'r/C'ru
C'rO/C'ru
RCR'p
2,62
11,81
0,38
0,08
0,30
Paste Datlab Graphs
0
0
1,0
C
C r
rO
C Cru
ru
R
A
0,
5
1,
0
1,
5
2,
0
2,
5
3,
0
3,
5
14,
0
4,
5
5,
0
5,
5
6,
0
6,
5
7,
0
7,
5
8,
0
8,
5
9,
0
O2 Concentration (F) [nmol/ml]
Cr
Mark
Date
Unit
Start
Stop
N Points
(E)
O2 Flow per cells (E)
O2 Concentration (F)
O2 Flow per cells (F)
Block Temp.
Barom. Pressure
Peltier Power
pX (E)
Slope X (E)
pX (F)
Slope X (F)
nmol/ml
pmol/(s*Mill)
nmol/ml
pmol/(s*Mill)
°C
kPa
%
V
Unit
V
Unit
01-Cr
00:15:33
00:18:27
87
130,61
25,81
134,29
24,97
37,00
86,76
-39,90
-9,99
0
-9,99
0
Paste Datlab Mark Statistics
Figure A1. Datlab analysis: “Mark Statistics-F2” are copied to clipboard and pasted into
the Excel template “O2k-DatLabAnalysis_TIP2k.xls” for each chamber (Chambers E
and F; of the third Oxygraph-2k used in the workshop). The bar graphs and respiratory
control ratios are then displayed automatically. The corresponding Oxygraph-2k traces
(DatLab graphs) for the two chambers are copied to clipboard and pasted into the
DatLab-Excel file.
OROBOROS INSTRUMENTS
O2k-Protocols
MiPNet10.04
Cell respiration and coupling control
14
Supplement B. Instrumental background test
22,94
a'+a°
87,60
b'+b°
200
4
2
120
0
80
-2
O2 Slope uncorr. (E) [pmol/(s*ml)]
160
O2 Concentration (E) [nmol/ml]
Mark
01
02
2005-04-10 EF-01.DLD
Averages Unit
J°1
J°2
Start
00:37:28 01:11:23
Stop
00:45:23 01:16:58
N Points
238
167
O2 Concentration
nmol/ml (E)163,69
84,12
X O2 Slope uncorr.
pmol/(s*ml)
(E) 1,92
0,18
O2 Concentration
nmol/ml(F) 163,33
81,47
O2 Slope uncorr.
pmol/(s*ml)
(F)
1,92
0,04
Block Temp.
°C
37,00
37,00
Barom. Pressure
kPa
87,60
87,60
Peltier Power
%
-38,59
-38,44
pX (E)
V
-9,99
-9,99
Slope X (E)Unit
0,00
0,00
pX (F)
V
-9,99
-9,99
Slope X (F)Unit
0,00
0,00
4
y = 0,0218x - 1,6521
40
Background O2 flux [pmol.s-1.cm-3]
Instrumental Background
Date
Chamber
E
Run
1
2005-04-10 EF-01.DLD
Calib. C
7,6092
0,0192
Backgr. C
-1,28
0,0232
Paste DatLab graph here, reduce to width 15 cm
3
y = 0,0218x - 1,6521
2
1
0
-1
-2
-4
0
00:00:00
00:17:43
00:35:25
Close
00:53:08
Range [hh:mm:ss]: 1:46:15
01:10:50
J°1
01:28:33
J°2
-3
01:46:15
J°3
Close
Cytc
AscorbatTMPD
0
50
Comments:
150
200
22,46
a'+a°
Mark
01
02
2005-04-10 EF-01.DLD
Averages Unit
J°1
J°2
Start
00:37:56 01:12:47
Stop
00:45:23 01:18:21
N Points
224
167
O2 Concentration
nmol/ml(E) 163,66
84,09
O2 Slope uncorr.
pmol/(s*ml)
(E)
1,94
0,27
O2 Concentration
nmol/ml (F)163,31
81,46
X O2 Slope uncorr.
pmol/(s*ml)
(F) 1,93
0,15
Block Temp.
°C
37,00
37,00
Barom. Pressure
kPa
87,60
87,60
Peltier Power
%
-38,61
-38,46
pX (E)
V
-9,99
-9,99
Slope X (E)Unit
0
0
pX (F)
V
-9,99
-9,99
Slope X (F)Unit
0
0
4
y = 0,0273x - 2,4242
87,60
b'+b°
200
4
2
120
0
80
-2
40
O2 Slope uncorr. (F) [pmol/(s*ml)]
160
Background O2 flux [pmol.s-1.cm-3]
Instrumental Background
Date
Chamber
F
Run
1
2005-04-10 EF-01.DLD
Calib. D
7,7646
0,0122
Backgr. D
-1,63
0,0270
Paste DatLab graph here, reduce to width 15 cm
O2 Concentration (F) [nmol/ml]
100
O2 concentration, c O2 [µM]
3
y = 0,0273x - 2,4242
2
1
0
-1
-2
-4
0
00:00:00
00:17:43
00:35:25
Close
00:53:08
Range [hh:mm:ss]: 1:46:15
01:10:50
J°1
01:28:33
J°2
Comments:
01:46:15
J°3
Close
-3
Cytc Ascorbat
TMPD
0
50
100
150
200
O2 concentration, c O2 [µM]
Datlab [O2] and JO2 traces
J°O2/ cO2 regression
Datlab Mark Statistics
Figure B1. Instrumental background of the Oxygraph-2k with a 2 ml chamber volume,
using the table sheet “Template O2k-Background” in the file “O2k-Background.xls”.
The O2k-Chamber is closed without sample, and after
stabilization for 10 min, oxygen consumption is
obtained of the polarographic oxygen sensor at air
saturation (Fig. B1; first mark: J°1). Open the chamber
partially without removing the stopper (lift the stopper
by about 1 cm and use the O2k-spacer for reproducible
stopper position), to obtain a gas phase above the
stirred medium. Then purge argon (or nitrogen) gas
into this gas phase, using the O2k-gas injection syringe
with an adequately fitted needle inserted through the
capillary of the stopper, to reduce the oxygen
concentration in the gas phase and medium. When
oxygen concentration has dropped by about 45%, the
stoppers are gently closed again, avoiding any gas
bubbles trapped in the chamber. Flux stabilizes after an
undershoot (Fig. B1), and the second mark, J°2, is set
on the section of stable flux. Continue with one or two
more reduced oxygen levels (Fig. B1; third mark: J°3).
The marks can be renamed automatically from a
template (Marks\Names; template “O2kBackground”).
The linear regression is automatically displayed in
DatLab and can be copied into the Excel template
“O2k-Background.xls”, table sheet “Template O2kBackground” (Fig. B1):
OROBOROS INSTRUMENTS
O2k-Protocols
MiPNet10.04
Cell respiration and coupling control

15
\2.Ok-Protcols\Files_Protocols\MiPNet14.06\O2kBackground.xls
Background oxygen flux is plotted as a function of
oxygen concentration with intercept, a° (-1.6 and -2.4
in Fig. B1), and slope, b° (0.0218 and 0.0273). These
values are used (1) to confirm proper function of the
respirometer (results are close to the default values of
−2 and 0.025), (2) to monitor the instrumental
characteristics over time (a° may become gradually or
suddenly more negative over weeks of experiments,
indicating an increase of a leak, possibly due to
defective O-rings on the stopper that must be
replaced),
and
(3)
for
real-time
instrumental
background correction of flux during respirometric
experiments in the corresponding O2k-Chambers.
OROBOROS INSTRUMENTS
O2k-Protocols