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Difference between revisions of "MiPNet14.06 Instrumental O2 background"

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{{OROBOROS header page name}}
{{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.
|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|100px|link=http://wiki.oroboros.at/images/6/65/MiPNet14.06_InstrumentalO2Background.pdf |Bioblast pdf]]
|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=2020-04-16
|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.''' » [http://www.bioblast.at/index.php/File:MiPNet14.06_InstrumentalO2Background.pdf Versions]
|abstract=Fasching M, Gnaiger E (2020) O2k Quality Control 2: Instrumental oxygen background correction and accuracy of oxygen flux. Mitochondr Physiol Network 14.6(08):1-16.
 
{{MiPNet pdf page linking to MitoPedia}}
'''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]]
 
O2k-Protocols SOP: O2k Quality Control 1 »[[MiPNet06.03 POS-calibration-SOP]]«
O2k-Manual »[[MiPNet19.18E O2 flux analysis]]«
:» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue |O2k-Catalogue]]
|keywords=Instrumental background
|mipnetlab=AT_Innsbruck_Oroboros
|mipnetlab=AT_Innsbruck_Oroboros
}}
}}
__TOC__
== Keywords ==
 
{{Template:Keywords: Chamber volume}}
[[File:Expand.png|right|45px |Click to expand or collaps]]
<div class="toccolours mw-collapsible mw-collapsed">
:::[[File:Questions.jpg|left|24px |Explanation of terms]] '''» Keywords'''
<div class="mw-collapsible-content">
::: '''Instrument'''
::::» [[O2k-FluoRespirometer]]
::::» [[DatLab]]
::::» [[O2k-Chamber]]
::::» [[MiPNet19.18E O2 flux analysis]] |O2k-Manual: O2 flux analysis]]
::::» [[MiPNet19.03 O2k-cleaning and ISS |O2k-Protocols SOP: O2k-cleaning]]
 
::: '''Concept'''
::::» [[Concentration]]
::::» [[High-resolution respirometry]]
::::» [[Instrumental background oxygen flux]]
::::» [[MiPNet14.06 Instrumental O2 background |O2k-Protocols SOP: Instrumental O2 background]]
::::» [[MitoFit Quality Control System]]
::::» [[O2k-SOP |O2k-Protocols SOP]]
::::» [[Oxygen |Oxygen, dioxygen, O<sub>2</sub>]]
</div>
</div>
 
{{Technical support integrated}}
 
::::* First step - O2k Quality Control 1: »[[MiPNet06.03 POS-calibration-SOP]]«
[[File:O2k-QCS.jpg|350px|O2k-QCS]]
 
::::* Second step - O2k Quality Control 2
 
[[Image:MiPNet14.06.jpg|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).]]
== Standard operating procedure ==
:::: If all quality control criteria of the O2 sensor test are met, the operator can be assured that the quality of the sensor signal is acceptable. Next, the quality of the O2k-Chamber assembly has to be tested, described in detail as an O2k-SOP:
:::: The '''O2k-chamber test''' provides quality control at an instrumental level beyond the O2 sensor test:
::::# [[O2k-Chamber]] not properly assembled or broken.
::::## [[OroboPOS-Holder]] not properly positioned.
::::## [[O2k-Chamber Holder]] not properly positioned; V- and O-rings not properly mounted ([[V-ring\30-35-4.5 mm]], [[O-ring\Viton\18x2 mm]]).
::::# [[Volume-Calibration Ring]] not properly positioned by chamber volume calibration.
::::# [[O-ring\Viton\12x1 mm]] injured and must be replaced on the stopper.
::::# [[Stopper\black PEEK\conical Shaft\central Port]] broken conical edge or O-ring not properly applied.
::::# [[OroboPOS-Seal Tip]] leaky.
::::# Experimental medium consumes oxygen due to microbial contamination.
 
::::* Next step - when measuring cytochrome ''c'' oxidase activity: Autoxidation of ascorbate and TMPD causes a [[MiPNet06.06_ChemicalBackground |chemical background oxygen flux]]. DatLab provides real-time correction for instrumental and chemical background.
 
 
== Trouble shooting ==
:::'''If specifications given in the [[MiPNet14.06 Instrumental O2 background |Instrumental O<sub>2</sub> background]] are not obtained''': check components for locating the problem.
::::# Check the stirring bars for any contamination.
::::# Check the stoppers for the quality of the O-rings and the conical edges.
::::# If no indications of a defect are observed, disassemble the O2k-chamber.
::::# Check the [[glass chamber]] for contamination or for broken edges.
::::# Clean the copper block of the O2k and reassemble the O2k-chamber.
::::# Reassemble and clean the chambers: [[MiPNet19.03 O2k-cleaning and ISS]].
::::# Perform an O2 sensor test and - if successful - an O2k-chamber test, using fresh incubation medium.
::::# If the problem with the instrumental O<sub>2</sub> background remains in one chamber, switch stoppers between chambers A and B.
::::# Perform an O2k-chamber test (the sensor test is not necessary).
::::# If the problem with the instrumental O<sub>2</sub> background remains in the same chamber, switch glass chambers between the left and right side of the O2k.
::::# Perform an O2 sensor test and - if successful - proceed with the O2k-chamber test.
::::# If the problem with the instrumental O<sub>2</sub> background remains in the same chamber, switch sensors between the left and right chamber.
 
 
=== Instrumental oxygen background test for permeabilized muscle fibres ===
 
:::# 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.
:::# 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.
:::# 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).
:::# 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.
:::# Increase O<sub>2</sub> with H<sub>2</sub>O<sub>2</sub> injection (c. 1 µl) to 450-500 µM, until the fibres are added, for equilibrating the instrument at high O<sub>2</sub>.
:::# 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 [[Talk:Closed_chamber|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<sup>-1</sup>·ml<sup>-1</sup>. The actual values should correspond very closely to the expected slope, i.e. within ± 1 pmol·s<sup>-1</sup>·ml<sup>-1</sup>.
::::* Values higher than 4-5 pmol·s<sup>-1</sup>·ml<sup>-1</sup> at 37 °C may therefore indicate a [[Talk:Biological_contamination|biological contamination]] in the chamber or in the medium.
::::* Lower values may indicate:
::::# 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.
::::# A large volume of medium collected in the receptacle of the stopper: siphon off any medium.
::::# A larger chamber volume: check [[O2k-Chamber volume calibration]].
::::# 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.
 
 
 
 
 
 
 
 
 
 
 
 
 
== Instrumental O2 DLP ==
 
Instrumental O2 [[DL-Protocols]] (DLP) are used for calibrations and instrumental quality control, typically without experimental sample in the incubation medium.
 
{| class="wikitable" border="1"
|-
! DL-Protocol
! Description
! DLP File
 
|-
| Instrumental O2 background TIP2k
| Instrumental O2 background with TIP2k including OroboPos test and two-point calibration
| [[Media:Instrumental O2 background_TIP2k.DLP|Instrumental O2 background TIP2k.DLP]]
|-
| Instrumental O2 background manual injections
| Instrumental O2 background with manual injections including OroboPos test and two-point calibration
| ''In progress''
|-
| Instrumental high O2 background TIP2k
| Instrumental high O2 background with TIP2k including OroboPos test and two-point calibration
| ''In progress''
|-
| Instrumental high O2 background manual injections
| Instrumental high O2 background with manual injections including OroboPos test and two-point calibration
| ''In progress''
|}  
 
A full list of DL-Protocols (Instrumental DL-Protocols and SUIT DL-Protocols) is displayed  in [[DL-Protocols]].
 
== DatLab-Analysis templates and DatLab Demo Files ==
 
[[File:2017-05-17 P2-01 Instrumental O2 background TIP2k.jpg|400px|left]]
 
:: '''DatLab 7:''' [[Media:O2 background.xlsx|Instrumental O2 background.xlsx]]
 
:: '''DatLab 6:''' [[Media:O2k-Background DL6.xlsx|O2k-Background.xlsx]]
 
:: '''DatLab 5:''' [[Media:O2k-Background DL5.xlsx|O2k-Background.xlsx]]
 
 
:: '''Demo file:'''
::: [[Media:MiPNet14.06 2014-07-24 P4-02 Instr-background.DLD|MiPNet14.06_2014-07-24_P4-02_Instr-background.DLD]] (norm. oxygen)
::: [[Media:MiPNet10.04 2014-02-20 P4-02 O2-calib high-O2.DLD|MiPNet10.04_2014-02-20_P4-02_ O2-calib high-O2.DLD]] (high oxygen)
 
== 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. - [[Gnaiger_1995_J_Bioenerg_Biomembr |»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. - [[Gnaiger_2001_Respir_Physiol |»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«]]




'''Acknowledgements'''
[[File:Template NextGen-O2k.jpg|left|400px|link=NextGen-O2k]]
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{{Labeling
{{Labeling
|area=Respiration, Instruments;methods
|area=Respiration, Instruments;methods
|instruments=Oxygraph-2k, O2k-Protocol
|instruments=Oxygraph-2k, O2k-Manual
|additional=O2k-SOP, DatLab
|additional=DatLab, O2k-SOP, DL7, System
}}
}}
[[Category:OroboPedia]]

Revision as of 17:23, 18 October 2020


                  


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MiPNet14.06 Instrumental O2 background


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O2k-Manual
O2k Quality Control 2: Instrumental oxygen background correction and accuracy of oxygen flux.

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Oroboros (2020-04-16) Mitochondr Physiol Network

Abstract: Fasching M, Gnaiger E (2020) O2k Quality Control 2: Instrumental oxygen background correction and accuracy of oxygen flux. Mitochondr Physiol Network 14.6(08):1-16.

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