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| | {{Template:OROBOROS support page name}} |
| {{Publication | | {{Publication |
| |title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] Instrumental oxygen background correction, accuracy of oxygen flux and SOP. [[Media:MiPNet14.06 InstrumentalO2Background.pdf |»Bioblast pdf«]] | | |title=[[Image:O2k-Manual.jpg|right|70px|link=O2k-Manual|O2k-Manual]] O2k Quality Control 2: Instrumental oxygen background correction and accuracy of oxygen flux. |
| |info=[http://www.bioblast.at/index.php/File:MiPNet14.06_InstrumentalO2Background.pdf Versions]
| | |info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/6/65/MiPNet14.06_InstrumentalO2Background.pdf |Bioblast pdf]] » [http://www.bioblast.at/index.php/File:MiPNet14.06_InstrumentalO2Background.pdf Versions] |
| |authors=OROBOROS | | |authors=Oroboros |
| |year=2015-04-24 | | |year=2023-10-19 |
| |journal=Mitochondr Physiol Network | | |journal=Mitochondr Physiol Network |
| |abstract=Fasching M, Gnaiger E (2015) Instrumental oxygen background correction, accuracy of oxygen flux and SOP. Mitochondr Physiol Network 14.6(04):1-8. | | |abstract=Timón-Gómez A, Grings M, Baglivo E, Schmitt S, Gnaiger E (2023) O2k Quality Control 2: Instrumental oxygen background correction and accuracy of oxygen flux. Mitochondr Physiol Network 14.6(09):1-16. |
| | {{MiPNet pdf page linking to MitoPedia}} |
| | :» Product: [[O2k-FluoRespirometer]], [[Oroboros O2k-Catalogue |O2k-Catalogue]] |
| | |mipnetlab=AT_Innsbruck_Oroboros |
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| Correction for the instrumental oxygen 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 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 routine correction.
| | == Keywords == |
| | {{Template:Keywords: Chamber volume}} |
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| '''O2k-Protocol for instrumental background correction and accuracy of oxygen flux'''
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| For calibration of the polarographic oxygen sensor (POS) and for measurement of instrumental background oxygen consumption, incubation medium without biological sample is added into the O2k 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 O2k 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.
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| :» Product: [[Oxygraph-2k|OROBOROS O2k]], [[O2k-Catalogue_OROBOROS| O2k-Catalogue]]
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| |keywords=Instrumental background
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| |mipnetlab=AT_Innsbruck_OROBOROS}}
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| {{Labeling
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| |area=Respiration, Instruments;methods
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| |instruments=Oxygraph-2k, Protocol
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| |additional=O2k-SOP, DatLab}}
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| [[Image:MiPNet14.06.jpg|centre|frame|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).]]
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| Autoxidation of ascorbate and TMPD causes a [http://bioblast.at/images/0/0b/MiPNet06.06_ChemicalBackground.pdf chemical background oxygen flux]. DatLab provides on-line correction for instrumental and chemical background.
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| >> [http://www.oroboros.at/?backgroundcorrection#c3005 Excel Templates and DatLab-Demo Files]
| | '''Acknowledgements''' |
| | [[File:Template NextGen-O2k.jpg|left|400px|link=NextGen-O2k]] |
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| | | {{Labeling |
| | | |area=Respiration, Instruments;methods |
| == Instrumental oxygen background test for permeabilized muscle fibres ==
| | |instruments=Oxygraph-2k, O2k-Manual |
| | | |additional=DatLab, O2k-SOP, DL7, System, MitoFit2022QC |
| === Extended instrumental oxygen background test ===
| | }} |
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| # 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.
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| # 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.
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| # Instrumental background: After 20 min, open the chamber and allow O<sub>2</sub> to drop to c. 350 µM, close for 20 min, open and drop O<sub>2</sub> to c. 250 µM (this should be the lowest experimental O<sub>2</sub> concentration).
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| # Increase O<sub>2</sub> with H<sub>2</sub>O<sub>2</sub> injection (c. 2 µl) to 400 µM, measure for 15-20 min instrumental background, simulating a re-oxygenation during the experiment.
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| # Increase O<sub>2</sub> with H<sub>2</sub>O<sub>2</sub> injection (c. 1 µl) to 450 µM, until the fibres are added, for equilibrating the instrument at high O<sub>2</sub>.
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| # Addition of permeabilized fibres into the O2k-Chamber: >> [[Permeabilized muscle fibres]].
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| == Flux in closed chamber near air saturation (in preparation) ==
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| {{Technical support integrated}}
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| {{#set: Technical service=O2 signal|Technical service=Performance test}}
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| The oxygen flux 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 (Details tab/ Oxygen consumption by POS), see 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<sup>-1</sup>·ml<sup>-1</sup>. The actual values should be within ± 1 pmol/(s·ml).
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| Values higher than 4-5 pmol·s<sup>-1</sup>·ml<sup>-1</sup> at 37° may therefore indicate a [[biological contamination]] in the chamber or in the medium (but double check the theoretical value from the Details tab). Lower values may indicate:
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| * a higher chamber volume: check and if necessary repeat [[chamber volume calibration]]
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| * air bubbles: remove air bubbles
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| * if a multi-sensor stopper with multiple bores is used: liquid on top of the stopper creating an exchange of liquid between the chamber volume and the outside: aspirate excess liquid from top of stopper
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| For different fluxes in the left and right chamber (with or without sample) see also [[Different O2 fluxes in left and right chamber]]
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| == Further information ==
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| '''References'''
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| [[Gnaiger_1995_J_Bioenerg_Biomembr| 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.]]
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| [[Gnaiger_2001_Respir_Physiol| Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128:277-97.]]
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| [[Gnaiger_2008_POS| 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.]]
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| :>> [http://www.oroboros.at/?BackgroundCorrection '''Excel Templates and DatLab-Demo Files''']
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| [[Category:OroboPedia]]
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