International Workshops on High-Resolution Respirometry
IOC59
2010 October 1 - 6: O2k-Workshop Obergurgl, Austria, Introductory O2k-Workshop on High-Resolution Respirometry - Satellite to the 7th Conference on Mitochondrial Physiology, MiP2010: Sep 27 - Oct 01.
IOC60
2010 December 11 - 16, O2k-Workshop Schroecken, Austria, Introductory O2k-Workshop on High-Resolution Respirometry
Table of contents
General Information
The O2k-Course on High-Resolution Respirometry (HRR) includes a basic introduction to the O2k, overview presentations on HRR and its applications in mitochondrial physiology. Specific emphasis is placed on hands-on practical sessions with the instrument and the software DatLab 4.3, to introduce participants into the technical and operational details of the method. An overview is given on the O2k-MultiSensor system, demonstrating our new TPP+ electrodes for the measurement of mt-membrane potential.
The workshop programme is extended by lectures and experiments on substrate-uncoupler-inhibitor titration (SUIT) protocols. Short presentations of 'Hot topics in Mitochondrial Physiology' extend the discussions on respiratory protocols.
Experienced lecturers guide small working groups step-by-step through the approach of high-resolution respirometry. Five Oxygraph-2k (with accessories and PCs with DatLab 4; four TIP-2k) are available to provide everybody with a do-it-yourself opportunity for both hardware and software.
Preliminary Programme - Introductory O2k-WorkshopDay 1
Arrival
18:30 Welcome reception
19:00 Dinner
21:00-21:20 Introduction of participants and their research interests.
Day 2
Principles of HRR - with a little help from a friend: the O2k-Manual.
8:30 – 09:30 The O2k and HRR: Introduction and oxygen calibration of the polarographic oxygen sensors (OROBoPOS).
09:30 – 10:30 Hands-on: Oxygen sensor calibration with DatLab 4.3
10:30 Coffee break
11:00 – 12:00 Erich Gnaiger: Experimental protocols for substrate-uncoupler-inhibitor titrations (SUIT protocols): An introduction.
12:00 - 14:00 Lunch break - exercise
14:30 -16:15 Demo experiment – the Oxygraph-2k and on-line DatLab analysis. Yeast as a HRR-model.
16:15 Coffee
17:45 – 18:45 Hands-on: Experiment with the Oxygraph-2k (five O2k - 10 chambers) and on-line DatLab analysis.
19:00 Dinner
21:00 Discussion of results, protocol, DatLab analysis.
Day 3
08:30 – 09:30 Erich Gnaiger: Instrumental background - Introduction.
09:30 – 12:00 Hands-on (five groups): Oxygen calibration and instrumental background test with the Oxygraph-2k - Washing and filling the O2k chambers with experimental media; air calibration; instrumental background competition, DatLab background analysis.
A. Instrumental background test for experiments with cells and isolated
mitochondria, from air saturation to zero oxygen concentration, with
automatic TIP-2k titration.
B. Instrumental background test for experiments with permeabilized muscle
fibres, in the high-oxygen range of 400 to 200 µM. Manual titration of
hydrogen peroxide into MiR06 (MiR05 with catalase).
12:00 Lunch break – sports
16:00 Coffee,tea
16:30 – 17:15 Background analysis – Summary.
17:15 – 17:45 DatLab 4.3 – An overview.
17:45 – 18:45 Hands-on (five groups): Instrumental background analysis.
19:00 Dinner
21:00 Hot topics: MiPNet Sessions
Day 4
08:15 Parallel group sessions - Introduction
Setup POS Service DatLab Analysis
-------------------------- -------------------------------------
08:30 – 09:15 Gr. 1 Gr. 2 Gr. 3 Gr. 4 Gr. 5
09:15 – 10:00 Gr. 5 Gr. 1 Gr. 2 Gr. 3 Gr. 4
10:00 Coffee
Setup POS Service DatLab Analysis; general topics
-------------------------- -------------------------------------
10:30 - 11:15 Gr. 4 Gr. 5 Gr. 1 Gr. 2 Gr. 3
11:15 – 12:00 Gr. 3 Gr. 4 Gr. 5 Gr. 1 Gr. 2
12:00 – 12:45 Gr. 2 Gr. 3 Gr. 4 Gr. 5 Gr. 1
13:00 Lunch - sports
16:00 Coffee/ tea
16:30 - 17:30 Working groups: Elaborate answers to the ‘Questions for the O2k-Course’
17:30 - 18:45 Presentation of ‘Answers for the O2k-Course’ – Trouble shooting
19:00 Dinner
Day 5
08:30 - 10:30 Erich Gnaiger: Experimental protocols. a) Phosphorylation control protocol with intact cells: ROUTINE – LEAK – ETS. b) Diagnostic multiple SUIT protocols with mt preparations.
10:30 Coffee break
11:00 - 12:00 Kathrin Renner-Sattler: Trouble shooting in HRR
12:00 Lunch break
14:00 - 15:00 Dominik Pesta: Permeabilized muscle fibres – preparation and HRR. MiPNet 3.
15:00 - 16:00 Open topics:
A. O2k MultiSensor overview.
B. O2k and advanced DatLab 4.3 features.
C. Practise DatLab 4.3 with demo data.
D. Operation of the Titration-Injection microPump – TIP-2k.
16:00 Coffee
16:30 – 17:00 Discussion-Summary–Conclusions
17:15 Special announcement
Farewell party
Day 5
Early morning: Departure
Contents O2k-Workshop: Overview on High-Resolution Respirometry
Introduction: Mitochondrial and cellular respiratory physiology; new challenges for high instrumental performance.
High-resolution respirometry; what makes the difference? Presentation of the OROBOROS Oxygraph-2k
- Optimum system design - the OROBOROS Oxygraph-2k.
DatLab 4: on-line recording of oxygen concentration and flux; linear slope versus oxygen flux as a function of time. DatLab 4: the specialized software for high-resolution respirometry; high-resolution calibrations. Low oxygen and measurement of cellular oxygen consumption – pushing the limits of detection.
OROBOROS Oxygraph-2k and TIP-2k: On-line instrumental performance
MultiSensor applications: TPP+, pH, NO
Conceptual and methodological advantages of measurement at physiological low levels of oxygen.
Polarographic oxygen sensor (POS) and O2k service
Cleaning of anode and cathode.
Electrolyte and membrane application.
Oxygraph-2k and TIP-2k: instrumental maintenance.
Contact
O2k-Workshop:
Erich Gnaiger, PhD
OROBOROS® INSTRUMENTS GmbH (Corp.)
high-resolution respirometry
Schöpfstrasse 18
A-6020 Innsbruck, Austria
Tel: +43 512 566796
Fax: +43 512 566796 20
E-mail: erich.gnaiger@oroboros.at
MITOFOOD COST Action Number FA0602
Coordinator: Dr. Jaap Keijer
Head Food Bioactives
RIKILT - Institute of Food Safety
Wageningen University
Bornsesteeg 45
PO Box 230
6700 AE Wageningen, Netherlands
jaap.keijer@wur.nl
