MiPNet19.18E O2 flux analysis

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MiPNet19.18E O2 flux analysis


Publications in the MiPMap
O2k-Manual
Oxygen flux analysis: DatLab real-time.

» Bioblast pdf

OROBOROS (2016-08-08) Mitochondr Physiol Network

Abstract: Gnaiger E (2016) Oxygen flux analysis: DatLab real-time. Mitochondr Physiol Network 19.18(E04):49-60. » Versions


O2k-Manual: DatLab sets a novel standard in high-resolution respirometry, for on-line analysis of oxygen flux measured in the OROBOROS O2k, and of other signals obtained in the O2k-MultiSensor System. Oxygen flux can be plotted with or without O2kbackground correction, is instantaneously expressed per mass of sample or per number of cells. Various sections on the plot of oxygen flux are marked, and corresponding average values are viewed in a table which can be simply exported into Excel or SigmaPlot. Instrumental and experimental parameters are summarized in a protocol, which can be printed or saved as a pdf file. These new features provide the basis for combining high-resolution with instant and user-friendly analysis.


A demonstration experiment, performed during an O2k Workshop on high-resolution respirometry, is used as an example for application of DatLab and DatLab-Excel templates MiPNet08.09. All analyses can be performed real-time or disconnected from the O2k.

» DatLab demo files and Excel templates: MiPNet14.06 Instrumental O2 background
» Product: DatLab 7, O2k, O2k-Catalogue

HRR-DatLab O2k-Network Lab: AT_Innsbruck_OROBOROS


Labels: MiParea: Respiration, Instruments;methods  HRR: Oxygraph-2k, O2k-Manual, O2k-Protocol Additional: O2k-Core, DatLab 


OROBOROS support

O2k-technical support

This information is part of O2k-technical support and open innovation.


Contents


Questions.jpg

DatLab plots of O2 flux per mass or flow per cell (per unit sample) remain shown per volume

  • Problem: After selecting a plot of the O2 flux per mass or flow per cell (per unit sample or the), the plot in the graph remains shown as flux per volume. Neither selecting the plot manually in 'Graph - Select Plots' (in the menu) nor choosing the appropriate layout from the menu 'Layout' helps.
  1. Cause
    1. Sample Concentration\Amount inside the 'Edit Experiment' window (menu 'Experiment - Edit') equal to 0
    2. The information of the Sample Unit\Concentration\Amount inside the 'Edit Experiment' window (menu 'Experiment - Edit') hasn't been saved yet. Although the values inside this window are taken from the last experiment (measured with this DatLab installation, where the 'Save' button inside the 'Edit Experiment' has been pressed) and displayed at the beginning of a next experiments, they are not automatically saved. - This is only relevant up to DatLab version 6.
  2. Solution
    1. Enter the Concentration\Amount different from 0
    2. Inside the 'Edit Experiment' window (menu 'Experiment - Edit') review (or enter) the sample information (Unit\Concentration\Amount), click on the 'Save' button - then the appropriate plot can be selected (if the Concentration\Amount is not equal to 0).
~ Capek Ondrej 15:24, 2 April 2015 (CEST)


Questions.jpg

Fluxes are different in left and right chamber

  • Question: In our experiments we get consistently higher fluxes in chamber A as compared to chamber B.
  • Solutions
  1. To exclude instrumental causes please follow the procedure described in the section Calibration and quality control of the OroboPOS (O2k-SOP) in MiPNet06.03 POS-calibration-SOP.
  2. Possible instrumental and protocol related causes for fluxes differing between chambers:
    1. Wrong O2 calibration in one chamber, see MiPNet06.03 POS-calibration-SOP.
    2. Wrong chamber volume calibration: The total amount of oxygen in the chamber with the smaller volume will be smaller. Therefore, the O2 concentration will drop faster resulting in a higher flux, see MiPNet19.18A O2k-start.
    3. Wrong background parameters causeing an O2 concentration-dependent offset to the flux, the value of the offset will be independent of the absolute value of the flux. E.g. a constant offset of 10 pmol / s ml at ca 100 µM O2 concentration and 20 pmol/s ml at 300 µM O2 concentration could be caused by wrong background parameters, but a difference of always e.g. 20% at very different absolute values of the flux can not be caused by wrong background parameters. Check the background parameters (perform an instrumental O2 background experiment). See MiPNet14.06 Instrumental O2 background.
    4. Sample injection: One filling of a syringe is used to inject the sample into several chambers. Sedimentation starts to occur immediately and more sample will be injected into the first chamber than in any subsequent chambers. A remedy we apply is to fill the syringe with sample sufficient for 3 chambers and inject the first aliquot back to the sample stock solution before quickly injecting the rest into the chambers. Independently of this we recommend to randomize chamber assignment to prevent systematic errors.
    5. Hydrophobic inhibitors, see MiPNet19.03 O2k-cleaning and ISS.
    6. Biological contamination, see Biological contamination.
    7. Hardware defects: While hardware defects (sensors, electronics,..) can obviously have many negative effects (noise signal, slow response, no signal,..) it is difficult to see how a real hardware defect can cause a systematic error in flux calculation once a correct calibration of the O2 sensor (at air saturation and at zero oxygen) was obtained.
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