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MitoPedia O2k and high-resolution respirometry: O2k-Open Support
What are potential causes for different O2 fluxes in the left and right chamber?
- 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 POS-calibration.
- Possible instrumental and protocol related causes for flux differences between chambers:
- Incorrect O2 calibration in one chamber, see POS-calibration.
- Incorrect 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 O2k-Series H: Start.
- Incorrect background parameters: incorrect background parameters will cause 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 constant difference of 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 Instrumental O2 background.
- 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 randomizing the assignment of the chambers to prevent systematic errors.
- Hydrophobic inhibitors, see MiPNet19.03 O2k-cleaning and ISS.
- Biological contamination, see Biological contamination.