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| | {{Template:OROBOROS support page name}} |
| {{Publication | | {{Publication |
| |title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] O2k Quality Control 2: Instrumental oxygen background correction and accuracy of oxygen flux. [[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=[[File:PDF.jpg|50px|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] | | |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=2016-02-09 | | |year=2023-10-19 |
| |journal=Mitochondr Physiol Network | | |journal=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. | | |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}} |
| '''[[O2k-SOP |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.
| | :» Product: [[O2k-FluoRespirometer]], [[Oroboros O2k-Catalogue |O2k-Catalogue]] |
| | | |mipnetlab=AT_Innsbruck_Oroboros |
| :» O2k-Protocols SOP: O2k Quality Control 1 »[[MiPNet06.03 POS-Calibration-SOP]]« | |
| :» O2k-Manual »[[MiPNet19.18E O2 Flux Analysis]]«
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| :» Product: [[Oxygraph-2k|OROBOROS O2k]], [[OROBOROS O2k-Catalogue |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, O2k-Protocol
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| |additional=O2k-SOP, DatLab
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| }} | | }} |
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| __TOC__
| | == Keywords == |
| | {{Template:Keywords: Chamber volume}} |
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| {{Technical support integrated}}
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| [[Image:MiPNet14.06.jpg|right|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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| 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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