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Difference between revisions of "Oxygen flux - instrumental background"

From Bioblast
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After an air calibration at high oxygen (~400 µM) the chamber won't exponentially oxygenate and won't reach the same level as the other chamber (which contains the same media and was oxygenated with the same syringe). Sometimes the air calibration shows a relatively low POS signal voltage around 1.4 (usually very close to 2.0) but not always. The background calibration is correct as is the gain, stir speed, and temperature.  
After an air calibration at high oxygen (~400 µM) the chamber won't exponentially oxygenate and won't reach the same level as the other chamber (which contains the same media and was oxygenated with the same syringe). Sometimes the air calibration shows a relatively low POS signal voltage around 1.4 (usually very close to 2.0) but not always. The background calibration is correct as is the gain, stir speed, and temperature.  
It happens to our O2ks - but different chambers on random different days - and not consistently the same chamber or same O2k. It is usually the same user who processes our muscle biopsies, but I'm pretty sure it's some setting or mistake we're making and not a problem with the actual POS or program. Any help or suggestions are appreciated!
It happens to our O2ks - but different chambers on random different days - and not consistently the same chamber or same O2k. It is usually the same user who processes our muscle biopsies, but I'm pretty sure it's some setting or mistake we're making and not a problem with the actual POS or program. Any help or suggestions are appreciated!
[[File:Ticket2020011631000015 — High O2 Question_A.jpg|400px|center]]
[[File:Ticket2020011631000015 — High O2 Question_A.jpg|400px|center]]
[[File:Ticket2020011631000015 — High O2 Question_B.jpg|400px|center]]
[[File:Ticket2020011631000015 — High O2 Question_B.jpg|400px|center]]


'''Answer:'''
'''Answer:'''


I assume that your observed issue is related to one or more of the following experimental points:
::::: I assume that your observed issue is related to one or more of the following experimental points:


1) Do you always open the stoppers using the stopper-spacer ( https://www.bioblast.at/index.php/Stopper-Spacer) making sure that both chambers have the same fixed gas phase?
::::# Do you always open the stoppers using the stopper-spacer ( https://www.bioblast.at/index.php/Stopper-Spacer) making sure that both chambers have the same fixed gas phase?
2) Do you check that you have a clear gas phase when you look inside the chamber and not several 'bubbles'?
::::# Do you check that you have a clear gas phase when you look inside the chamber and not several 'bubbles'?
3) Do you inject O2 in the order of  1<sup>st</sup> the 'good chamber' followed by 'bad chamber'? Do you always take fresh O<sub>2</sub> before injection between each chamber or do you use the remaining O<sub>2</sub> from the 1<sup>st</sup> injection?
::::# Do you inject O2 in the order of  1<sup>st</sup> the 'good chamber' followed by 'bad chamber'? Do you always take fresh O<sub>2</sub> before injection between each chamber or do you use the remaining O<sub>2</sub> from the 1<sup>st</sup> injection?
4) Do you inject the same amount of O2?
::::# Do you inject the same amount of O<sub>2</sub>?


Additionally, when you inject O<sub>2</sub> for the first time in both chambers there is an identical O<sub>2</sub> concentration slope (although the increase in chamber B isslower). In this situation you need to wait for the desired O<sub>2</sub> concentration longer in chamber A than B (which is just fine, such O<sub>2</sub> increase can be slow due to many points mentioned from 1-4). Instead you have injected several times more O<sub>2</sub> without waiting long enough for reaching the desired concentration. In fact, that O<sub>2</sub> concentration is continuously increasing in chamber B even after the stirrer test.
Additionally, when you inject O<sub>2</sub> for the first time in both chambers there is an identical O<sub>2</sub> concentration slope (although the increase in chamber B is slower). In this situation you need to wait for the desired O<sub>2</sub> concentration longer in chamber A than B (which is just fine, such O<sub>2</sub> increase can be slow due to many points mentioned from 1-4. Instead you have injected several times more O<sub>2</sub> without waiting long enough for reaching the desired concentration. In fact, that O<sub>2</sub> concentration is continuously increasing in chamber B even after the stirrer test.
We recommend to close the chamber immediately after reaching the target concentration (450 to 550 µ for pfi), which displaces the gas phase and stops the equilibration process.
We recommend to close the chamber immediately after reaching the target concentration (450 to 550 µM for pfi), which displaces the gas phase and stops the equilibration process.


=== Zero oxygen concentration reached but 'Instrumental_O2_background_TIP2k.DLP' is not finished  ===
=== Zero oxygen concentration reached but 'Instrumental_O2_background_TIP2k.DLP' is not finished  ===

Revision as of 11:50, 6 February 2020


high-resolution terminology - matching measurements at high-resolution


Oxygen flux - instrumental background

Description

Instrumental background oxygen flux, J°O2, in a respirometer is due to oxygen consumption by the POS, and oxygen diffusion into or out of the aqueous medium in the O2k-Chamber. It is a property of the instrumental system, measured in the range of experimental oxygen levels by a standardized instrumental background test. The oxygen regime from air saturation towards zero oxygen is applied generally in experiments with isolated mitochondria and living or permeabilized cells. To overcome oxygen diffusion limitation in permeabilized fibers and homogenates, an elevated oxygen regime is applied, requiring instrumental background test in the same range of elevated oxygen.

Abbreviation: J°O2

Reference: MiPNet14.06 Instrumental O2 background; Gnaiger_2008_POS; Gnaiger_2001_RespPhysiol

» See MiPNet14.06 Instrumental O2 background for experimental details of the instrumental background test, and for downloading the Excel template for analyzing instrumental background experiments.
» Compare Chemical background correction of oxygen flux.

Template NextGen-O2k.jpg


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




O2k-QCS

O2 slope in the closed and open O2k-Chamber

Instrumental background correction eliminates errors by systemic flux compensation, automatically performed by DatLab. If no experimental background test has been performed, the system default values are used, which are a°=-2.0 pmol/(s·mL) for the intercept at zero oxygen concentration, and b°=0.025 for the slope of background flux as a function of oxygen concentration.
Automatic correction for the instrumental background oxygen flux is an essential standard in high resolution respirometry. At the same time an instrumental background experiment is the ultimate test for instrumental performance, evaluating chamber performance after completion of all elements of the Oxygen sensor test. The instrumental background oxygen flux measured at air saturation should reflect the theoretically predicted volume-specific oxygen consumption by the oxygen sensor. The actual agreement using experimental respiration medium provides at the same time a test that excludes microbial contamination of the medium or serves to evaluate any autoxidation processes in newly tested experimental media.
In an open chamber of the O2k the liquid phase in the chamber (aqueous medium) is in equilibrium with the atmosphere. All oxygen consumed by the polarographic oxygen sensor (POS) is immediately replaced from the atmosphere. The oxygen signal therefore has to be constant and the (negative) time derivative of the oxygen signal, called "O2 slope uncorr." in DatLab, has to be zero. The background corrected oxygen flux is meaningless for the open chamber situation. This is because the background correction at air saturation subtracts the consumption of oxygen by the sensor from the negative slope, when diffusion into and out of the chamber is zero at air saturation. Therefore, the background-corrected oxygen flux in the open chamber at air saturation is shown as a negative value. To avoid this apparent artefact, the "O2 slope uncorr." is selected to be shown while the chamber is open. Only Graph Layouts that display "O2 slope uncorr." are suitable for assessing the stability of the oxygen signal when the chamber is open. Such Layouts are:
  • 01 Calibration show Temp
  • 02 Calibration - Background
The observation of a zero flux with an open chamber is an important performance parameter. It indicates that thermal stability and equilibrium of oxygen between the gas and aqueous phases have been reached. Therefore no experiment should be started before a zero "O2 slope uncorr." has been reached with an open chamber. The suggested criterion for signal stability is a "O2 slope uncorr." between -1 pmol/(s mL) and + 1 pmol/(s mL).


O2k-QCS

  • 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).


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:
  1. O2k-Chamber not properly assembled or broken.
    1. OroboPOS-Holder not properly positioned.
    2. 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).
  2. Volume-Calibration Ring not properly positioned by chamber volume calibration.
  3. O-ring\Viton\12x1 mm injured and must be replaced on the stopper.
  4. Stopper\black PEEK\conical Shaft\central Port broken conical edge or O-ring not properly applied.
  5. OroboPOS-Seal Tip leaky.
  6. Experimental medium consumes oxygen due to microbial contamination.
  • 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 fibers

  1. While biopsy sampling and fiber 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 fibers are added, for equilibrating the instrument at high O2.
  6. Addition of permeabilized fibers into the O2k-Chamber: » Permeabilized muscle fibers.

Instrumental O2 DLP

Instrumental O2 DL-Protocols (DLP) are used for calibrations and instrumental quality control, typically without experimental sample in the incubation medium. All Instrumental DL-Protocols available can be found in C:\DatLab\DL-Protocols\Instrumental\ (default directory after installation of DatLab).
A general description of DL-Protocols (Instrumental DL-Protocols and SUIT DL-Protocols) is displayed in DL-Protocols.


Instrumental O2 background representative traces

2017-05-17 P2-01 Instrumental O2 background TIP2k.jpg

Troubleshooting

If specifications given in the Instrumental O2 background are not obtained

Check components for locating the problem.
  1. Check the stirring bars for any contamination.
  2. Check the stoppers for the quality of the O-rings and the conical edges.
  3. If no indications of a defect are observed, disassemble the O2k-chamber.
  4. Check the glass chamber for contamination or for broken edges.
  5. Clean the copper block of the O2k and reassemble the O2k-chamber.
  6. Reassemble and clean the chambers: MiPNet19.03 O2k-cleaning and ISS.
  7. Perform an O2 sensor test and - if successful - an O2k-chamber test, using fresh incubation medium.
  8. If the problem with the instrumental O2 background remains in one chamber, switch stoppers between chambers A and B.
  9. Perform an O2k-chamber test (the sensor test is not necessary).
  10. If the problem with the instrumental O2 background remains in the same chamber, switch glass chambers between the left and right side of the O2k.
  11. Perform an O2 sensor test and - if successful - proceed with the O2k-chamber test.
  12. If the problem with the instrumental O2 background remains in the same chamber, switch sensors between the left and right chamber.

Oxygen concentration during air calibration at high oxygen not reproducible between chambers

  • Anonymous user; Heinrich Heine University Düsseldorf

Question: After an air calibration at high oxygen (~400 µM) the chamber won't exponentially oxygenate and won't reach the same level as the other chamber (which contains the same media and was oxygenated with the same syringe). Sometimes the air calibration shows a relatively low POS signal voltage around 1.4 (usually very close to 2.0) but not always. The background calibration is correct as is the gain, stir speed, and temperature. It happens to our O2ks - but different chambers on random different days - and not consistently the same chamber or same O2k. It is usually the same user who processes our muscle biopsies, but I'm pretty sure it's some setting or mistake we're making and not a problem with the actual POS or program. Any help or suggestions are appreciated!

Answer:

I assume that your observed issue is related to one or more of the following experimental points:
  1. Do you always open the stoppers using the stopper-spacer ( https://www.bioblast.at/index.php/Stopper-Spacer) making sure that both chambers have the same fixed gas phase?
  2. Do you check that you have a clear gas phase when you look inside the chamber and not several 'bubbles'?
  3. Do you inject O2 in the order of 1st the 'good chamber' followed by 'bad chamber'? Do you always take fresh O2 before injection between each chamber or do you use the remaining O2 from the 1st injection?
  4. Do you inject the same amount of O2?

Additionally, when you inject O2 for the first time in both chambers there is an identical O2 concentration slope (although the increase in chamber B is slower). In this situation you need to wait for the desired O2 concentration longer in chamber A than B (which is just fine, such O2 increase can be slow due to many points mentioned from 1-4. Instead you have injected several times more O2 without waiting long enough for reaching the desired concentration. In fact, that O2 concentration is continuously increasing in chamber B even after the stirrer test. We recommend to close the chamber immediately after reaching the target concentration (450 to 550 µM for pfi), which displaces the gas phase and stops the equilibration process.

Zero oxygen concentration reached but 'Instrumental_O2_background_TIP2k.DLP' is not finished


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. - »Bioblast link«
  • Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. - »Bioblast link«
Click to expand or collaps


MitoPedia concepts: MiP concept 


MitoPedia methods: Respirometry 


MitoPedia O2k and high-resolution respirometry: DatLab, Oroboros QM