MiPNet14.06 Instrumental O2 background

                

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MiPNet14.06 Instrumental O2 background

O2k Quality Control 2: Instrumental oxygen background correction and accuracy of oxygen flux. »Bioblast pdf«

»  » Versions

OROBOROS (2016-02-09) Mitochondr Physiol Network

Abstract: Fasching M, Gnaiger E (2016) O2k Quality Control 2: Instrumental oxygen background correction and accuracy of oxygen flux. Mitochondr Physiol Network 14.6(05):1-8.

O2k-Protocols SOP: Correction for instrumental background oxygen flux is a standard in high-resolution respirometry, automatically performed by DatLab. Background measurements provide a test of instrument function. In the OROBOROS O2k, 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 compliance to standard operating procedures (O2k-SOP)described in this chapter as part of the MitoFit Quality Control System. This is part 2 of O2k Quality Control.

» O2k-Protocols SOP: O2k Quality Control 1 »MiPNet06.03 POS-Calibration-SOP«

» O2k-Manual »MiPNet19.18E O2 Flux Analysis«

» Product: OROBOROS O2k, O2k-Catalogue

• Keywords: Instrumental background

• O2k-Network Lab: AT_Innsbruck_OROBOROS

Labels: MiParea: Respiration, Instruments;methods 

HRR: Oxygraph-2k, O2k-Protocol 

O2k-SOP, DatLab 

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MitoPedia O2k and high-resolution respirometry: O2k-Open Support 

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• First step - O2k Quality Control 1: »MiPNet06.03 POS-Calibration-SOP«

• Second step - O2k Quality Control 2

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).

• Next step - when measuring cytochrome c oxidase activity: Autoxidation of ascorbate and TMPD causes a chemical background oxygen flux. DatLab provides real-time correction for instrumental and chemical background.

Instrumental oxygen background test for permeabilized muscle fibres

1. While biopsy sampling and fibre preparation proceed: Perform 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 450-500 µM with oxygen gas (Syringe\60 ml\Gas-Injection), close and after two to three min perform a stirrer test (using the automatic stirrer test function of DatLab). 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 450-500 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.

Apparent oxygen flux in closed chamber near air saturation with pure medium

The uncorrected slope of the oxygen signal over time in a closed chamber at air-calibrated oxygen concentration is an important control parameter. It reflects the consumption of oxygen by the polarographic oxygen sensor (POS). The theoretical value is calculated by DatLab in the O2 Calibration window (Supplement C in MiPNet06.03 POS-Calibration-SOP). The theoretical value at 37 °C (O2 slope uncorrected) is usually between 2 and 3 pmol·s^-1·ml^-1. The actual values should correspond very closely to the expected slope, i.e. within ± 1 pmol·s^-1·ml^-1.

• Values higher than 4-5 pmol·s^-1·ml^-1 at 37 °C may therefore indicate a biological contamination in the chamber or in the medium.
• Lower values may indicate:

1. Air bubbles in the closed chamber: switch on the internal illumination of the O2k and inspect the chamber through the front window. Remove any air bubbles.
2. A large volume of medium collected in the receptacle of the stopper: siphon off any medium.
3. A larger chamber volume: check chamber volume calibration.
4. If an O2k-MultiSensor stopper with multiple ports is used, it is particularly important to siphon off excess liquid from top of stopper. Any convection of liquid must be avoided, which otherwise results in an apparently leaky chamber.

Excel templates

» Excel Templates and DatLab-Demo Files

References

• Gnaiger E, Steinlechner-Maran R, Méndez G, Eberl T, Margreiter R (1995) Control of mitochondrial and cellular respiration by oxygen. J Bioenerg Biomembr 27:583-96. - »Bioblast link«
• Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128:277-97. - »Bioblast link«
• 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_2008_POS |»Bioblast link«]]