Oroboros Instruments Oxygraph-2k User manual

OROBOROS INSTRUMENTS O2k-Core Manual
Oxygraph-2k:
start
high-resolution
respirometry
Gnaiger E, Fasching M,
Gradl L, Gradl P
OROBOROS INSTRUMENTS Corp
high-resolution respirometry
Schöpfstr 18, A-6020 Innsbruck, Austria
Email: [e-mail]
www.oroboros.at
Section 1. OROBOROS USB-Stick and DatLab installation 7 Page
2. Unpack the Oxygraph-2k..............................8
3. Placing the Oxygraph-2k .............................8
4. Assembly of the Oxygraph-2k ......................8
4.1. Glass chamber and POS assembly ................9
4.2. Chamber volume calibration - O2k-SOP ....... 10
5. Start-up the Oxygraph-2k ......................... 11
6. Start-up DatLab ....................................... 12
6.1. O2k-MultiSensor –Configuration ................ 12
6.2. O2k-MultiSensor - Control [F7] .................. 12
6.3. Connect and Save DLD File ........................ 14
6.4. O2k-signal line .......................................... 14
6.5. O2k-status line ........................................ 15
7. Oxygraph control ...................................... 16
7.1. O2k control keys ...................................... 16
7.2. Control [F7] with the O2k connected .......... 16
7.3. Disconnect ............................................... 17
Supplement: DatLab 5 and O2k Series A-D ........S1
Overview: The OROBOROS Oxygraph-2k (O2k) provides a unique high-
resolution approach to the monitoring of cellular and mitochondrial
respiratory function. High-resolution respirometry (HRR) is indispensable at
low respiratory activities, kinetic transitions and low oxygen levels. High
resolution is required for analysis of pathological effects causing reduced
Mitochondrial Physiology Network 19.01(A01): 5-18 (2014) 2014 OROBOROS
A: www.bioblast.at/index.php/MiPNet19.01A_O2k-Start Version A01: 2014-03-06
Oxygraph-2k Manual
OROBOROS Oxygraph-2k
A

MiPNet19.01 A: O2k-Start 6
OROBOROS INSTRUMENTS OROBOROS Oxygraph-2k
respiration (mitochondrial and metabolic diseases, ischemia-reperfusion
injury, oxidative stress, apoptosis, aging). HRR is unique for analysis of
biopsies with limited amounts of sample (diagnosis of genetic and
acquired mitochondrial defects in pediatric patients, diabetes); cell
cultures with limited cell numbers or low activities; mutants with
diminished respiratory capacity; chemical oxidation rates and antioxidant
capacities; and respiratory measurements at low, physiological
intracellular oxygen levels and oxygen kinetics.
HRR with the OROBOROS O2k is based on a radically different
systems approach to polarographic measurement of oxygen dynamics.
HRR rests on tightly connected instrumental features and concepts
integrated by the DatLab software, providing real-time display of oxygen
concentration and respiration (oxygen flux). The mechanics, critical
selection of materials, and state-of-the-art electronics provide the basis of
signal stability of an optimized polarographic oxygen sensor (OroboPOS),
in an electronically controlled thermal environment with high temperature
stability. Calibration and instrumental control follow standardized
operating procedures (O2k-SOP) with automatic real-time analysis
supported by the DatLab software. Scientific support complements HRR.
Detailed laboratory O2k-Protocols on HRR provide step-by-step
instructions. Daily new O2k-Publications on applications of the OROBOROS
Oxygraph-2k focus on critical issues of mitochondrial physiology.
While the OROBOROS Oxygraph-2k provides the instrumental basis
for high-resolution respirometry, successful operation at high accuracy up
to the limit of detection depends on a professional application by the
technician, scientist or student. Participation in a hands-on introductory
O2k-Workshop is recommended to save time and gain from the
experience obtained over many years of application in various settings.
The OROBOROS Oxygraph-2k (O2k)
is the modular system for high-
resolution respirometry (HRR). The
O2k is the 2-chamber
high-resolution respirometer for
monitoring oxygen consumption with
small amounts of biological samples.
The modular O2k-concept yields a
high flexibility for HRR, with the
O2k-Core as the basis, and O2k-
Modules supported by the O2k-Core.
Available O2k-Modules are the
Titration-Injection microPump
(TIP2k) and the O2k-MultiSensor
Modules (Fluorometry; Ion
Selective Electrodes, ISE).

MiPNet19.01 A: O2k-Start 7
OROBOROS INSTRUMENTS O2k-Core Manual
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The O2k-Core is the experimental system complete for basic
high-resolution respirometry (HRR). The O2k-Core includes the O2k-Main
Unit with stainless steel housing, O2k-Assembly Kit, two OroboPOS
(polarographic oxygen sensors) and OroboPOS-Service Kit, the DatLab
software, the ISS-Integrated Suction System and the Titration Set. The
O2k-Core supports all add-on O2k-Modules of the Oxygraph-2k.
1. OROBOROS USB-stick and DatLab installation
A laptop or PC is required for data acquisition and
analysis using DatLab. Minimum configuration: Pentium
III, 256 MB, USB port, Windows XP, Windows 7.
Copy the entire folder “OROBOROS O2k-Course on
HRR” from the OROBOROS USB-Stick to your
computer. The ‘OROBOROS-FileFinder’is the tool to
find topics, such as all chapters of the O2k-Manual
and O2k-Protocols, with corresponding DatLab demo
files and templates for training with DatLab.
Open Access updates:
» www.oroboros.at/?O2k-Manual
» www.oroboros.at/?O2k-Protocols

MiPNet19.01 A: O2k-Start 8
OROBOROS INSTRUMENTS OROBOROS Oxygraph-2k
DL5Inst.exe: It is recommended that you exit all Windows
applications before running the DatLab Installation
program. In the OROBOROS-FileFinder’, click the
OROBOROS symbol or ‘O2k-DatLab Install’. Read the
short instruction windows, and proceed by clicking
Next. By default, DatLab is installed to "C:\DatLab".
Installation is quick and easy and generates this icon on
your screen: Oroboros DatLab for the control of the
Oxygraph-2k, TIP2k, data acquisition & analysis.
Drag the DatLab icon into an appropriate position on
your screen.
2. Unpack the Oxygraph-2k
After unpacking, store the original Box 1 (or Peli
Case) and packing material for any future
shipment of the Oxygraph-2k.
Box 1 (or the Peli Case) contains the O2k-Main Unit, including
the chamber holders in a mounted position, the holders
for the polarographic oxygen sensors (OroboPOS) and
the POS connectors (optionally the TIP2k syringes).
Box 2 contains the standard accessories [SA], the OROBOROS
USB-Stick incl. DatLab software and documentation,
and optional O2k-Modules and accessories.
3. Placing the Oxygraph-2k
The Oxygraph-2k requires a minimum of lab space
(0.5 m bench space) next to an external PC or laptop,
with sufficient space for the ISS, syringes and handling.
For maximum signal stability and optimal temperature
control, the O2k should be placed in a position in which
it is not exposed to direct sunlight and sudden changes
of room temperature. Keep the space below the
Oxygraph-2k bottom clear to prevent any interference
of ventilation beneath the O2k-Main Unit.
Avoid damage due to condensation. When placing the
O2k from a cold to warm environment, let it equilibrate
for at least 4 hours in the warm room before connecting
to the mains.
4. Assembly of the Oxygraph-2k
Uncharge yourself to avoid possible damage of the
electronics. Shortly connect to electrical ground or
simply touch the O2k metal housing (MiPNet14.01).
OroboPOS assembly:
O2k-Manual B » MiPNet19.01B_POS-Service

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OROBOROS INSTRUMENTS O2k-Core Manual
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Remove the POS connectors from the POS holders by
pressing the sleeve downwards against the bottom
plate of the POS connector. Unplug the cable, and
unscrew the chamber holder from the copper block.
The POS are shipped dry (without electrolyte) in a
Perspex housing in the OroboPOS-Service Kit. Before
assembling the Oxygraph-2k, apply electrolyte and
membrane, following the OroboPOS service instructions
in MiPNet19.01B. Clean the gold-plated pin and thread
of the POS connector, using a smooth cloth wetted with
distilled water, then with a cloth wetted with pure
ethanol (or methanol), finally with a dry cloth.
4.1. O2k-Glass chamber and OroboPOS assembly
O2k-SOP: Insert the glass chamber into the copper block of
the O2k-Main Unit, then add the PVDF stirrer bar.
1. Screw the POS holder [1] into a position
where the glass chamber with the angular cut
towards the POS holder is slightly lifted upwards.
2. Screw the chamber holder loosely to the
copper block (arrow [2]). Loosen the POS holder
stepwise (counterclockwise, not more than
successive quarter turns; arrow [1]) and follow
any downwards movement of the glass chamber
by screwing down the chamber holder (arrow
[2]). When a slight backwards movement of the
POS holder [1] cannot be followed by any further
downwards movement of the chamber holder [2], then
1
2

MiPNet19.01 A: O2k-Start 10
OROBOROS INSTRUMENTS OROBOROS Oxygraph-2k
3
4
5
4
screw the POS holder [1] clockwise (finger-tight), thus
securing the glass chamber in a fixed position.
3. Screw a POS head onto each POS connector (finger-
tight). Push a wetted POS seal tip (black gasket) over
the POS head (arrow [3]). Position the pore centrally
and do not strech the gasket.
4. Connect the POS connector cable to the O2k-Main
Unit, avoiding bending or torsions of the cable. Insert
the male plug of the cable [4] into the female plug next
to the control light of the stirrer. The red dot on the
male plug [4] has to face straight upwards when
inserting the plug. Each OroboPOS head can be used on
chamber A or B of the Oxygraph-2k. Note the POS
number (marked on the cylindrical body of each POS)
for each chamber (see Section 6.1).
5. The POS connector cable [4] must be in a relaxed,
undistorted position. Press the sleeve downwards
(arrow [5]), insert the POS with the black gasket
straight into the POS holder, and release the sleeve for
fixing the POS in its final position.
Do not rotate the POS connector after it is attached
to the POS holder in order to prevent damage of the
black gaskets. The gaskets provide a tight seal against
the glass chamber that has sharp edges.
4.2. O2k-Chamber volume calibration
O2k-SOP: DatLab must be installed before calibrating the
chamber volume. Connect the O2k to the mains
(Section 5), connect the O2k with DatLab (Section 6),
and make yourself familiar with the O2k control keys
(stirrer; Section 7.1). The standard chamber volume is
2.0 ml.
1. Dry the chamber. Remove all liquid
droplets from the glass chamber. Place a
dry PVDF stirrer into each chamber.
2. Add accurately 2.1 ml into the chamber
for calibration of a chamber volume of 2.0
ml (more accurately, the volume of the
stopper capillary is 0.085 ml or 85 µl when
a small meniscus of water is seen on top of
the capillary). The volume of the capillary
of the stopper (black PEEK) is not part of
the effective chamber volume. When titrating 2.1 ml,
insert the tip of the pipette to the wall of the glass
chamber to add the entire volume. Liquid must not be
lost between the top of the glass cylinder and the blue
POM chamber holder.

MiPNet19.01 A: O2k-Start 11
OROBOROS INSTRUMENTS O2k-Core Manual
A
3. Switch the stirrers on (Section 7.1).
4. Loosen the stopper fixation rings (A) and (B) with the
L-shaped Allen key (OroboPOS-Service Kit) and push
them slightly downwards in the direction of the O-rings.
5. Insert the stopper with dry capillary (the O-rings should
be moistened) and push it downwards until the gas
phase fully extrudes through the capillary of the
stopper, while the fixation ring is fully pushed onto the
chamber holder.
6. Gently push the stopper further downwards until the
capillary fills up and a small droplet appears on the top
of the capillary of the stopper. This marks the volume
calibration position.
7. The fixation rings (A) and (B) are fixed by gently
tightening the screws with the Allen key.
5. Start-up the Oxygraph-2k
Check the suitability of the delivered cable for your
power and plug in the mains power cable.
The O2k is delivered with one of three different types:
•US: O2k-Main Power Cable\120 V\US-CA, 21112-01
•Europe: O2k-Main Power Cable\230 V\Europe,
21111-01*
•Australia and New Zealand: O2k-Main Power
Cable\230 V\AU-NZ, 21113-01
*Note: Use O2k-Main Power Cable\230 V\Europe
(Hybrid German/French plug CEE7/7, Type E+F) only
for a compatible socket. Some Danish sockets will also
accept the CEE7/7 plugs, but there is no grounding
connection with these plugs without male ground pin.
Exchange for an appropriate Danish power cable.
Connect the O2k with the USB cable to a laptop or PC.

MiPNet19.01 A: O2k-Start 12
OROBOROS INSTRUMENTS OROBOROS Oxygraph-2k
Switch on the O2k on the rear (Mains on/off switch:
I/0). Now the four front control lights illuminate: The
outer left diode (MAINS) is green, and temperature is
regulated at 25 °C. The inner left and right diodes
(STIRRER) are red when the default stirrer setting is
OFF. The outer right diode (COMM) is yellow as long as
the O2k is not operationally connected to DatLab.
WGT Quality Control Seal: Do not open. An intact quality control seal
is required for warranty obligations related to the
electronics of the Oxygraph-2k.
6. Start-up DatLab
On your PC screen, click on the icon “Oroboros DatLab”
to start the program.
Either the O2k Control window opens automatically by
default, or open O2k-Control by pressing [F7].
With the USB cable connected,
select “USB Port” in the
Connection window. If several O2ks
are connected to one PC, the serial
numbers of all O2ks are shown to
select one. Press Cancel for
disconnected operation of DatLab.
6.1. O2k-MultiSensor - Configuration
OroboPOS #: The number of the polarographic oxygen sensor is
entered for chambers A and B. Usually, sensors should
not be switched between chambers, except for
troubleshooting. The individual sensor number is
included in the protocol for generating a database of
calibration values (MiPNet06.03).
Oxygraph control at start [On/Off]: Should be “On”
() if the PC is used with the Oxygraph-2k. Switch the
option to “Off” for a PC used separately for data
analysis.
Save Configuration: Click on Save configuration to save changes.
Subsequently, you may click on Cancel to close the
window, or proceed in the Control window. The settings
are also saved when immediately changing to the
Control window.
6.2. O2k-MultiSensor –Control [F7]
In the “O2k-MultiSensor” menu [F7], Control the
Oxygraph-2k operation mode. Temperature, gain

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OROBOROS INSTRUMENTS O2k-Core Manual
A
settings, polarization voltage, stirrer speed, data
recording interval and settings used upon connect
(stirrer power and light) are edited either step-by-step,
or by loading an O2k-Setup. The default is “O2k-
Setup 25 °C” or the O2k-Setup which was applied last
when the PC was connected to the Oxygraph-2k.
Settings of selected setups are displayed after Load
setup. Activate the displayed control settings by
clicking on the Connect to Oxygraph-2k button.
Block temperature [°C]: Click on the input window (double click to
overwrite all digits), and enter the block temperature of
your choice (range 2.0 to 47.0 °C).
Gain for oxygen sensor: Can be changed for signal amplification in
specific cases. The default is a gain of 1 V/µA; options
are 1, 2, 4, or 8 V/µA amplifications. In particular, use a
low gain if sections of the experiment are performed at
high oxygen above air saturation, when the maximum
voltage is close to 10 V or higher (offscale). Use a higher
gain at low signal output when the maximum voltage
obtained during an experiment is <3 V (relevant only at
low temperature).
Polarization voltage [mV]: The default for the POS is 800 mV,
which is maintained in all routine experiments. Any
changes affect the calibration. The voltage can be
modified in the range of 2,000 to +2,000 mV for
scanning the plateau region of the polarographic
oxygen sensor, or for different types of sensors.
Amp and pX: Settings for the Amp and pX panels are described in
MiPNet17.05 O2k-Fluorescence LED2-Module,
MiPNet15.05 O2k-NO Amp-Module, and MiPNet15.03
O2k-MultiSensor-ISE.
Stirrer speed [rpm]:The default is 750 rotations per minute (12.5
Hz), which may be varied between 300 and 900 rpm for
each chamber independently. The signal level and
signal stability increase with stirrer speed in the region
of low rpm, but uncoupling of the magnet occurs at
very high stirring speeds with the consequence of high
signal noise. Both chambers may be set at an identical
stirrer speed without electronic interference.
Data recording interval [s]: Can be set between 0.2 s and 10.0 s,
identical for both chambers. The default of 2 s is
optimal for most applications. Do not decrease the data
recording interval unless necessary, since at low data
recording intervals, the apparent noise of flux is
increased and real-time information is reduced.
Stirrer power [On/Off]: Toggle between On/Off to activate or
deactivate the stirrer after connection. Independent of
this On/Off setting, the left and right stirrers are

MiPNet19.01 A: O2k-Start 14
OROBOROS INSTRUMENTS OROBOROS Oxygraph-2k
switched on and off during the experiment by pressing
[F11] and [F12].
Light in chambers [On/Off]: Toggle between On/Off to switch the
light on or off after connection. Independent of this
On/Off setting, the light is switched on and off during
the experiment by pressing [F10].
Save setup: Save the Oxygraph control settings under a setup
name. To create a new O2k-Setup, edit the name in the
window, and click on the Save setup button.
O2k-Setup files can be renamed or deleted in the menu
Oxygraph \ Manage Setups.
Connect to Oxygraph-2k: Click on Connect to Oxygraph-2k.
6.3. Connect and save DLD file
In an automatically opened Save window, the default file
name has the form: 2014-02-12 P1-01.DLD
Year-Month-Day P#- (= ower-O2k P#) 0#
(=sequential number of the experiment). Extension DLD
for DatLab Data.
Select a subdirectory and edit the file name at this
stage. Then click on the Save button. The DLD file is
saved, and the communication is set up between DatLab
and the O2k. Data recording and saving is initiated
automatically.
Successful connection is checked on the ‘Comm’ diode of
the O2k Main unit, which changes to green, and in the
O2k status and signal lines at the bottom of the screen.
6.4. O2k signal line
Time [s]: Time of the experiment since connecting, displayed on
the X-axis as [hh:mm] or [hh:mm:ss].
O2(A) [V] and O2(B) [V]: Oxygen raw signal of chamber (A) and
(B), which is converted to oxygen concentration by
calibration, as displayed in the plot O2 concentration.
If the oxygen signal displays 9.99 V, the amplifier has
reached saturation. Immediately after polarization of the
sensors, the signal may be very high, declining slowly to
a lower steady-state value. If the signal remains at 9.99
V, press [F7] and reset the gain to a lower value. Click
O2k status line
O2k signal line
Time [s] O2 [V] Temp. [°C] Peltier power [%] pX [V] Amp [V]
Barometric pressure [kPa]
(A) and (B)
(A) and (B)