MiPNet14.06 Instrumental O2 background

From Bioblast
Jump to navigation Jump to search
Publications in the MiPMap
Fasching M, Gnaiger E (2009) Instrumental background correction and accuracy of oxygen flux. Mitochondr Physiol Network 14.6.

» Open Access-MiPNet14.06.pdf; Versions

O2k-Protocols contents

OROBOROS (2009) Mitochondr Physiol Network

Abstract: Correction for the instrumental background is a new and essential standard in high resolution respirometry, automatically performed by DatLab. Background measurements provide a test of instrument function. In the OROBOROS Oxygraph-2k, background corrections are usually within a few % of experimental flux over the entire experimental oxygen range. At minimum activities, however, even the small background effects become highly significant and require routine correction.

Instrumental background experiment, measuring oxygen flux without biological sample at four oxygen levels (left), and linear relation between instrumental background oxygen flux and oxygen concentration (right). Modified after: Gnaiger E. (2001).

Autoxidation of ascorbate and TMPD causes a chemical background oxygen flux. DatLab provides on-line correction for instrumental and chemical background.

Excel Templates and DatLab-Demo Files


O2k-Protocol for Instrumental Background Correction and Accuracy of Oxygen Flux

For calibration of the polarographic oxygen sensor (POS) and for measurement of instrumental background oxygen consumption, only incubation medium but no biological sample is added into the Oxygraph-2k chamber, at experimental conditions. In a closed chamber under these conditions, ideally oxygen concentration remains constant. In practice, however, instrumental background effects are caused by back-diffusion into the oxygraph chamber at low oxygen pressure, oxygen diffusion out of the oxygraph chamber at elevated oxygen levels, and oxygen consumption by the polarographic oxygen sensor (POS). Instrumental background interferes with accurate measurement of respiratory oxygen flux, if background effects remain undefined. Determination of instrumental background constitutes an important standard operating procedure (SOP) in high-resolution respirometry (HRR) Instrumental background oxygen flux is (i) minimized in the OROBOROS Oxygraph-2k by instrumental design and selection of appropriate materials. In addition, (ii) instrumenal background is routinely tested, and (iii) background correction of oxygen flux is applied automatically by DatLab.

>> O2k-Protocols:Overall contents
>> Product: OROBOROS Oxygraph-2k, O2k-Catalogue

Keywords: Instrumental background

O2k-Network Lab: AT_Innsbruck_OROBOROS


Labels: MiParea: Respiration, Instruments;methods 





HRR: Oxygraph-2k, Protocol"Protocol" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property. 

O2k-SOP, DatLab 

Instrumental background test for permeabilized muscle fibres

Extended instrumental background test

  1. While biopsy sampling and fibre preparation proceed: Air calibration in MiR06Cr, then close the chamber to evaluate instrumental background at air saturation (c. 10 min): This is a quality control of the medium, important under field conditions, where medium preservation (sterility) may be less controlled than in the lab.
  2. Elevate oxygen concentration to 450 µM with oxygen gas (Syringe\60 ml\Gas-Injection), close and after two to three min perform a stirrer test (the new DatLab has an automatic stirrer test function, with defined duriation of stopping the stirrer and automatic restart of stirring, 40 s stirrer stop may be optimum). This is important, since the OroboPOS may have a different response time at elevated oxygen concentration. If the response time increases dramatically, then the sensor may even show a non-linear response to oxygen concentration at high oxygen levels.
  3. Instrumental background: After 20 min, open the chamber and allow O2 to drop to c. 350 µM, close for 20 min, open and drop O2 to c. 250 µM (this should be the lowest experimental O2 concentration).
  4. Increase O2 with H2O2 injection (c. 2 µl) to 400 µM, measure for 15-20 min instrumental background, simulating a re-oxygenation during the experiment.
  5. Increase O2 with H2O2 injection (c. 1 µl) to 450 µM, until the fibres are added, for equilibrating the instrument at high O2.
  6. Addition of permeabilized fibres into the O2k-Chamber: >> Permeabilized muscle fibres


Further information

References

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 (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128: 277-297.

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.


>> Intrumental Excel Templates and DatLab-Demo Files
>> MiPNet10.07 is integrated in MiPNet14.06.
  • Version 2: 2011-12-04
  • Version 1: 2009