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Different O2 fluxes in left and right chamber

From Bioblast

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Question: In our experiments we get consistently higher fluxes in chamber A as compared to chamber B.

Answer: 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.

Possible instrumental and protocol related causes for fluxes differing between chambers:

  • Wrong O2 calibration in one chamber, see MiPNet06.03 POS-Calibration-SOP.
  • 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.
  • Wrong background parameters: wrong background parameters will cause a 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 Β΅M O2 concentration and 20 pmol/s ml at 300 Β΅M 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. In any case, it is advisable to check the background parameters (perform an instrumental O2 background experiment). See MiPNet14.06 InstrumentalBackground.
  • Sample injection: Scenario: 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.
  • Hydrophobic inhibitors, see MiPNet19.03 O2k-cleaning and ISS.
  • Biological contamination, see Biological contamination.

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. Fasching Mario 09:49, 7 November 2014 (CET)