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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. When the Amplex UltraRed assay is performed in the presence of exogenously added superoxide dismutase, O2•- will be dismutated to H2O2, which will then be measured in the assay. 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

Are the calculations equivalent for MiR05-Kit and self-made MiR05?

The calculations of the chemical background fluorescence are the same. You need to use the H2O2 flux analysis with DatLab 7.4 template which corresponds to the MiR05-Kit Lot number that you use. If you use homemade MiR05 or do not add DTPA, we recommend calculating the chemical background fluorescence according to: How to Analyse with DatLab 7.4_2

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 calibrations are needed because the sensitivity of the Amplex UltraRed assay towards H2O2 changes. This change may be due to: 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

Is the sensitivity always around 1 V/µM or does it vary depending on sample type and fluorescence intensity?

The sensitivity value and its change over time may be dependent 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). In MiR05-Kit the sensitivity is usually between 1 and 2 V/µM, but using a typical KCl-based buffer the sensitivity is frequently higher than 2.5 V/µM. After addition of the biological sample, the sensitivity usually decreases owing to the radical scavenging properties of the sample. The sensitivity might be also dependent of the correct preparation and titration of the H2O2 calibration solution (it should be prepared every day freshly) and on how precisely the components of the Amplex UltraRed assay are titrated, therefore, the calibration before the sample addition is considered as a quality control step. The fluorescence intensity of the fluorescence sensors can directly influence the chemical background flux of the Amplex UltraRed assay, which might have an effect on the sensitivity. For more information, see: Komlodi 2018 Methods Mol Biol and Krumschnabel 2015 Methods Mol Biol

What does RET stand for?

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

Can I directly use NADH as substrate?

No, you cannot use NADH as a NADH-linked substrate for mitochondrial respiration since NADH is not permeable through the mitochondrial inner membrane. For more information, see: Nicotinamide adenine dinucleotide