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O2k-Webinar Q&A


2020-07-23 O2k-Applications: H₂O₂ production by Dr. Tímea Komlódi

How to export the raw data from DatLab into Excel spreadsheets for analysis?

Our O2 flux analysis with DatLab 7.4 video on how to set marks and how to analyze the data is available on our website. The H2O2 flux analysis video will be available soon. All the O2k-Videosupport can be found here: O2k-Videosupport

Why cannot we use liver homogenate to monitor H2O2 flux?

Liver homogenate cannot be used to monitor H2O2 flux, because of the optical properties of the cytosol. For further information, see: 2015 Biomolecules

How to interpret H2O2 fluxes in permeabilized fibers in relation to the high oxygen regime used?

In permeabilized fibers (pfi), ROS production may be artificially increased by high O2 pressures, but high O2 concentrations are needed to avoid O2 limitation. Consequently, pfi may not be an optimum model for studies of ROS production. Further information can be found here: High oxygen in permeabilized fibers versus oxygen limitation under hypoxia

What is the difference between Amplex UltraRed assay and the use of probes like MitoSOX?

Amplex UltraRed assay is specific for H2O2, while MitoSOX is specific for O2•- but not for other reactive oxygen species or reactive nitrogen species. For more information, see: MitoSOX and Amplex UltraRed.

I have a specific question on the sensitivity correction in the H2O2 flux analysis with DatLab 7.4 file provided by Oroboros: was this correction developed only for isolated mitochondria, or does it work for all sample types (e.g. permeabilized fibers)? Specifically, the "Plot equation for the calculation of the correction factors for H2O2 production.

The H2O2 flux analysis with DatLab 7.4 template provided with the DatLab 7.4 installation can be used in experiments performed with MiR05-Kit (or MiR05) as mitochondrial respiration medium, and there is no restriction to sample preparation, therefore, it can be used with all sample types if the measurement is conducted in MiR05-Kit or MiR05. More detailed information can be found in: MiPNet24.10 H2O2 flux analysis

Do we have to perform H2O2 calibration of the Amplex UltraRed assay after each sample run? Why it is necessary to perform multiple calibrations?

We recommend performing H2O2 calibration of the Amplex UltraRed assay before sample addition (in DatLab 7.4: AmR calibration.DLP), after sample addition and multiple times during the experiment (in DatLab 7.4: see DL-protocols for AmR, https://www.bioblast.at/index.php/MitoPedia:_SUIT#SUIT_A_FluoRespirometry:_recommended_protocols). Multiple calibration is needed because the sensitivity of the Amplex UltraRed assay towards H2O2 changes. This change may be the function of: 1) experimental time, 2) changes in the optical properties, 3) radical scavenging capacity owing to the sample, and 4) concentration of the accumulating resorufin (UltroxRed in the case of Amplex UltraRed). For further information, see: Komlodi 2018 Methods Mol Biol

What does RET stand for?

RET stands for reverse electron flow. For further information, see: Reverse electron flow from CII to CI

Can I directly use NADH as substrate?

No, you cannot, NADH is not permeable through the mitochondrial inner membrane. For more information, see: Nicotinamide adenine dinucleotide