Oroboros Instruments O2k-FluoRespirometer User Manual

Oroboros Instruments High-Resolution FluoRespirometry

O2k-FluoRespirometer manual

Mitochondrial Physiology Network 22.11(01):1-16 (2017)

Version 01: 2017-07-21 O2k-Series H DatLab 7 2017 Oroboros

wiki.oroboros.at/index.php/MiPNet22.11_O2k-FluoRespirometer_manual

O2k-FluoRespirometer
manual

Gnaiger E

1,2

, Sigl R1, Mészáros A1,

Capek O1, Gradl L3, Gradl P4

Oroboros Instruments

Schöpfstr 18, A-6020 Innsbruck, Austria
Email: [e-mail]

www.oroboros.at

Mitochondria and Cell Research

Section 1. Unpack the O2k .......................................... 3 Page

2. Oroboros USB-flash drive and DatLab ............ 3

3. Electric and lab space requirements ............... 4

4. Start the O2k .............................................. 6

5. Assembly of the O2k .................................... 6

6. Start DatLab ............................................... 9

7. O2k control ............................................... 14

Supplement A: Videosupport ............................. S1

Supplement B: O2k-Chamber assembly .............. S2

Supplement C: O2k-Chamber volume calibration . S3

Overview

The Oroboros O2k-FluoRespirometer provides a unique high-resolution
approach to the monitoring of cellular and mitochondrial respiratory function.
High-Resolution FluoRespirometry (HRFR) is indispensable for application of
SUIT protocols, at low respiratory activities, kinetic transitions, and controlled
oxygen levels. High-resolution is required for analysis of pathological effects
causing reduced respiration (mitochondrial and metabolic diseases,
ischemia-reperfusion injury, oxidative stress, apoptosis, aging). HRFR is
unique for simultaneous measurement of respiration and fluorometric
signals (H2O2 flux, mt-membrane potential, Ca2+, ADP-ATP
phosphorylation), 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 oxygen kinetic measurements at low,
physiological intracellular oxygen levels.

O2k-FluoRespirometer

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Oroboros Instruments Mitochondria and Cell Research

HRFR with the Oroboros O2k-FluoRespirometer is based on a radically
different systems approach to polarographic measurement of oxygen
dynamics. HRFR rests on tightly connected instrumental features and
concepts integrated by the DatLab 7 software, providing real-time display
of oxygen concentration and respiration (oxygen flux), and a fluorometric
signal and its time derivative (slope). 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) and

Smart Fluo-Sensors in an electronically controlled thermal environment

with high temperature stability. Calibration and quality control follow
standardized operating procedures (O2k-SOP) with automatic real-time
analysis supported by DatLab. Scientific support complements HRFR.
Detailed laboratory O2k-Protocols provide step-by-step instructions,
integrated as DL-Protocols into DatLab 7. Numerous new O2k-

Publications on applications of the Oroboros O2k focus on critical issues

of mitochondrial physiology.

While the Oroboros O2k-FluoRespirometer provides the instrumental
basis for High-Resolution FluoRespirometry, 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 O2k is the modular
2-chamber system for High­Resolution FluoRespirometry
(HRFR). O2k-FluoRespirometer:

simultaneous monitoring of oxygen
consumption and fluorescence with
small amounts of samples (isolated
mitochondria to permeabilized tissues
and cells). Included: O2k-Titration
Set, Titration-Injection microPump
(TIP2k), Integrated Suction System
(ISS). Add-on O2k-Modules: high
flexibility to expand the spectrum in
HRFR.

O2k-Manual user information

PLEASE STUDY THIS MANUAL

» wiki.oroboros.at/index.php/MiPNet07.08_User_information

» wiki.oroboros.at/index.php/O2k-Manual

MiPNet22.11 O2k-FluoRespirometer manual 3

Oroboros Instruments High-Resolution FluoRespirometry

The O2k-FluoRespirometer is the complete system for
High-Resolution FluoRespirometry (HRFR), including the O2k-Main

Unit with stainless steel housing, O2k-Assembly Kit, 2+1 OroboPOS (1

spare oxygen sensor), OroboPOS-Service Kit, 4 Smart Fluo-Sensors, DatLab
software (DatLab), ISS-Integrated Suction System, Titration-Injection
microPump (TIP2k) and O2k-Titration Set.

1. Unpack the O2k-FluoRespirometer

Box 1 (or Peli Case) contains the O2k-Main Unit with the O2k-

Chambers in a finally mounted position, the OroboPOS-

Connectors without polarographic oxygen sensors

(OroboPOS) and the TIP2k syringes.
After unpacking, store the original Box 1 (or Peli

Case) and packing material for any future
shipment of the O2k-Main Unit.

Box 2 contains standard accessories [SA], the Oroboros USB-

flash drive and accessories.

2. Oroboros USB-flash drive and DatLab

A laptop or PC is required for data acquisition and

analysis using DatLab. Minimum configuration: Intel­Core-2 or equivalent CPU, 2GB RAM, USB port, Windows

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Oroboros Instruments Mitochondria and Cell Research

XP or later Windows versions. Recommended: Intel i5 or
equivalent CPU, 4GB RAM, Windows 7, SSD.

Copy the folder “Oroboros O2k-Course on HRFR”
from the Oroboros USB-flash drive to your computer.
The ‘Oroboros-FileFinder’ is the tool to find topics and
open the files. Updates of the O2k-Manual and O2k-

Protocols are posted on our website.

DL7Install.exe It is recommended that you exit all computer

applications before running the DatLab installation

programme. In the ‘Oroboros-FileFinder’, L click
the Oroboros symbol or ‘O2k-DatLab install’.

Read the short instruction windows, and proceed
by L Next. By default, DatLab is installed to
"C:\DatLab".

 Installation is quick and easy and generates this icon on

your desktop: Oroboros DatLab for control of the O2k,
TIP2k, data acquisition & analysis.

Running DL7 For data safety reasons and optimal DatLab

performance, it is strongly recommend to operate your
DatLab files, DL-Protocols and DatLab templates on the
PC‘s local hard drive (HDD).

MitoPedia-O2k is integral to the O2k-Manual, where specifically

advanced sections on High-Resolution FluoRespirometry
are included:

» wiki.oroboros.at/index.php/MitoPedia:_O2k_hardware

» wiki.oroboros.at/index.php/MitoPedia:_DatLab

3. Electric and lab space requirements

Max. power input: 120 W

Current at 100-120 V: max. 1.0 A
Current at 220-240 V: max. 0.5 A
Power consumtion during standard operation (when
block temperature is stable at 37 °C and ambient
temperature of 22 °C): approx. 24 W

 Check the suitability of the delivered cable for the mains

power (230\120 V) and socket. The O2k is delivered with
one of three different cable types:

EU: O2k-Main Power Cable\230 V\Europe, 21111-01

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Oroboros Instruments High-Resolution FluoRespirometry

Note: Use O2k-Main Power Cable\230 V\Europe (Hybrid

German/French plug CEE7/7, Type E+F) only for a
compatible socket.

DK: 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.

CH: A different main power cable or adapter is required.

US: O2k-Main Power Cable\120 V\US-CA, 21112-01
AU/NZ: O2k-Main Power Cable\230 V\AU-NZ, 21113-01

The O2k requires a minimum of lab space (0.5 m bench
space) next to an external PC or laptop, with sufficient
space for the ISS, O2k-Titration Set 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 on the table below
the O2k 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 h in the warm room before connecting to
the mains.

Rear of the O2k-FluoRespirometer.
New features are the USB hub 2.0 with 4 external ports,
a plug for an external Pt1000 thermistor, and an optional
extension through an RS485 serial port.

WGT quality control seal: Do not open. An intact quality control seal

is required for warranty obligations related to the
electronics of the O2k.

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Oroboros Instruments Mitochondria and Cell Research

4. Start the O2k

Mains on Connect the O2k with the USB cable to a laptop or PC.

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 green and stirrer rotation is on. The outer
right diode (COMM) changes to yellow if a USB
connection is available, and to green after operational
connection to DatLab.

5. Assembly of the O2k-FluoRespirometer

 Uncharge yourself to avoid possible damage of the

electronics. Shortly connect to electrical ground or
simply touch the O2k steel housing.

5.1. Instrument overview

The O2k-FluoRespirometer is electronically equipped
with all required plugs for sensors: in addition to the O2

channel, the Amp channel is available
with separate Fluo plug in O2k-Series
H. The pX channel has a separate Ref
plug for the reference electrode.

5.2. O2k glass chamber assembly

The O2k-FluoRespirometer is shipped with

installed glass O2k-chambers, POS holders and
chamber holders, ready to use. The PVDF stirrer bar

has to be added to each chamber. The Volume­Calibration Rings on stoppers A and B for chamber A and
B are precalibrated for a working volume of 2.0 ml in
each chamber. Check for the labels A/B, since stoppers
A/B must be matched with chambers A/B.

For maintenance or replacement of the glass chambers

and subsequent volume calibration refer to Supplement

B and C.