O2k-Open Support alert - 2020
- Currently Answered Questions from the »Oroboros Ecosystem Agenda«
||Why it is necessary to perform multiple H2O2 calibrations when using the Amplex UltraRed (AmR) assay?|
The sensitivity to H2O2 of the Amplex UltraRed assay changes during the experiment, thus requires multiple calibrations. These sensitivity changes may be the function of: 1) experimental time, 2) changes in the optical properties of the respiration medium due to sample/chemical titration, 3) the radical scavenging capacity of the sample, and 4) the concentration of accumulating resorufin (UltroxRed in the case of Amplex UltraRed). Therefore, it is recommended to perform H2O2 calibration before sample addition, after sample addition and multiple times during the experiment.
- »Komlodi 2018 Methods Mol Biol«
The DLPs for AmR SUIT protocols already come with multiple steps of H2O2 calibrations implemented.
- » SUITbrowser«
- communicated by »Komlodi Timea« and »Cardoso Luiza«
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||What do you do if values, symbols, and/or units are not correctly displayed in the DatLab software?|
It is possible to retrieve the values, symbols and/or correct units by updating the Windows control panel settings.
- » Troubleshooting for problems with text in DatLab«
||How can you compare respirometric experiments using an internal normalization?|
Flux control ratios (FCR) are independent of external measurements (e.g. protein determination, citrate synthase activity) and therefore can be used to analyze data when other normalization methods cannot be applied. Learn more about how to use this option to assess qualitative changes of mitochondrial respiratory control with DatLab and the “O2 analysis template DL7.4”.
- »Flux control ratio«
Read more: Gnaiger Erich et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1 »Bioblast link«
||Did you know that you can apply instrumental O2 background test parameters (i.e., intercept and slope) from an existing file in DatLab? |
In the DatLab file, you can copy the Instrumental O2 background test parameters directly via the ‘Copy from file’ option in the ‘Slope configuration’ window.
For more information, see:
- »Copy from file«
||Did you know that instabilities of the slope of the oxygen signal (O2 slope neg.) can be prevented by the proper use of TIP2k-Filter Papers during an instrumental O2 background test?|
- »Unstable 'O2 slope neg.'« and »TIP2k Filter Papers«
||Did you know that experimental artefacts can occur in the Amplex UltraRed assay after injection of chemicals that interfere with the fluorescence signal?
- » Injection artefact«
||Is the noise seen in the slope of the oxygen signal (O2 slope neg.) within recommended values?|
- An 'O2 slope neg.' within ± 2 pmol∙s−1∙mL−1 is optimal (± 4 pmol∙s−1∙mL−1 is acceptable) at a data recording interval of 2 s and 40 data points selected for calculation of the slope.
- » Air and zero calibration«
||Do you know how the "switch components" approach can help you find what is causing your O2k to not reach the quality control specifications?|
Do not despair if specifications given in the O2k-Manuals are not obtained. See how to troubleshoot on your own.
- »Switch components approach«
||Do you know that a humid O2k-environment and/or liquid spillage may lead to a high raw POS signal [V]?|
See how to clean and dry the electrical connections of the OroboPOS-Connector.
- »Cleaning the electrical connections«
|| Do you know how to avoid underestimating/overestimating respiratory fluxes? Measuring flux at steady states with high-resolution respirometry is the key to obtaining correct values. |
Learn more about steady states including performance and mark setting for data analysis.
- »Steady state«
||Do you set marks correctly in the instrumental O2 background test when planning to apply the background correction to a SUIT protocol with a high O2 regime?|
The range of O2 concentrations selected in the instrumental background test should correspond to the range of experimental O2 concentrations.
- » O2 Background correction - troubleshooting«
||Did you know that some substrates alone cannot maintain a stable mitochondrial respiratory flux in OXPHOS?|
Unstable respiratory fluxes in OXPHOS might be due to substrates limitation.
- » Pyruvate - troubleshooting«
||Why are the oxygen levels different between chambers prior to my experimental run?|
After switching on the O2k, air calibration has to be performed; otherwise the displayed concentrations may deviate from accurate concentrations.
- » Oxygen calibration - troubleshooting«
||Did you know that you can edit the background parameters in the 'O2 analysis template DL7.4?|
If you did not apply a finally evaluated O2 background correction to your experimental DatLab file, you can edit the O2 background parameters directly in the O2 analysis template DL7.4 for completion of Oxygen flux analysis - DatLab 7.4.
- »SUIT: Browse DL-Protocols and templates«
||Do you want to search the best SUIT protocol for your research questions?|
- Use the SUITbrowser: »SUITbrowser«
||Do you know that you can use the Titration-Injection microPump (TIP2k) to perform an instrumental O2 background test when using the O2k-sV-Module?|
(1) By adjusting the dithionite concentration to 2.5 mM, the instrumental O2 background test can be performed with the "BG Feedback" settings. - »Dithionite«
(2) By adjusting the TIP2k setup with specific settings for the O2k-sV-Module, the instrumental O2 background test can be performed using the O2k-sV-Module. - »O2k-sV-Module Manual«
||Do you change the slope smoothing to 20 while running experiments with biological sample?
While running your experiment, especially during uncoupler titrations, it is important to decrease the slope smoothing in order to have a faster visualization of the titrations effect on respiration.
- »Slope smoothing«
||Calibration parameters are transferred to experimental DatLab files
Save calibration information from selected Marks while DatLab is connected to the O2k during an instrumental calibration run. Air calibration and zero oxygen calibration can be performed and saved in a single DatLab file or in separate DatLab files.
- » Oxygen calibration - DatLab«
||Reproducibility committment in high-resolution respirometry: quality control in oxygen calibration
In the DatLab 7.4 software, new instrumental DL-Protocols guide the user automatically step-by-step, to calibrate the instrument accurately. Reproducibility is monitored and documented when following user-friendly standard operating procedures (SOP).
- »Oxygen calibration - DatLab«
||Do you check the O2 flux at the end of the air calibration with the O2k-chamber closed?
In DatLab 7.4, instrumental DL-Protocols guide you automatically towards closing the O2k-chamber after air calibration, to monitor "O2 slope neg." (negative) as a test of any biological contamination which would increase oxygen consumption in the absence of a biological sample.
- »Oxygen calibration - DatLab«
||Do you set mark ‘R1’ and complete the air calibration correctly?
Your air calibration is not effective until you set mark ‘R1’ and click on Calibrate and copy to clipboard in the O2 calibration window.
- »Oxygen calibration - DatLab«
||Do you write the serial number of the polarographic oxygen sensor (POS) in the ‘O2k configuration’ window?
Including the POS serial number in the ´O2k configuration´ window is important to keep a track record of the sensors used according to the O2k. This will be especially helpful if troubleshooting is needed.
- »O2k configuration«
||Do you perform O2k-Chamber volume calibration?
The volume calibration is essential to ensure that the chamber volume is correctly calibrated to its standard volume of 2 mL (or 0.5 mL for the O2k-sV-Module).
- »O2k-Chamber MitoPedia«
||Do you use the correct O2 solubility factor (FM) in the ‘O2 calibration’ window in DatLab?
The O2 solubility factor varies according to the medium used and influences the air calibration.
- »Oxygen solubility factor«
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