Description
Abbreviation: NS(GM)
Reference: A: simultaneous determination of O2 and H2O2 flux in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)-SUIT-018
SUIT number: D040_1GMS;2D;3Ama
O2k-Application: AmR
MitoPedia concepts:
SUIT protocol,
SUIT A,
Find
MitoPedia methods:
Fluorometry
- MitoPedia: SUIT
- SUIT-category: NS(GM)
- SUIT protocol pattern: linear 1GMS;2D-
SUIT-018 AmR mt D040 is a protocol to study simultaneously the oxygen dependence of O2 flux and H2O2 flux in mitochondrial preparations. In our experiments H2O2 flux shows linear dependence on oxygen concentration in LEAK, OXPHOS, ET states and in ROX state measured in MiR05-Kit, therefore, we recommend varying the oxygen level in the O2k-Chamber several times during the experiment. It is advisable to detect H2O2 flux in the same state either at high oxygen concentration (tissue hyperoxia; [O2]> ~100-110 Β΅M) and at low oxyen concentration (tissue normoxia; [O2]< ~70-80 Β΅M), or keep always the oxygen concentration in tissue normoxia. It is also recommended reducing the oxygen concentration in the O2k-Chamber before the sample addition. The oxygen concentration in the chamber can be decreased by injecting nitrogen with a syringe or increased by reoxygenation or by oxygen gas injection.
Communicated by Cecatto C, Iglesias-Gonzalez J, Komlodi T and Gnaiger E (last update 2020-06-17)
Representative traces
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
0DTPA |
| |||
0SOD |
| |||
0HRP |
| |||
0AmR |
|
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1GMS | GMSL(n) | NS | CI&CII | 1GMS
|
2D | GMSP | NS | CI&CII | 1GMS;2D
|
3Ama | ROX | 1GMS;2D;3Ama
|
Strengths and limitations
- + Simple protocol to evaluate the oxygen dependence of H2O2 production in LEAK and OXPHOS states.
- + Combination of N- and S-pathways is physiologically more relevant than using simple substrate to support a specific mitochondrial pathway.
- + Optionally, at the end of the protocol the effect of oxygen concentration can be studied in the ROX state (in the presence of Ama) both on the O2 and H2O2 fluxes.
- - It is advisable to adjust the optimal sample concentration in the O2k-Chamber beforehand in order to avoid unnecessary reoxygenations (high sample concentration) or to avoid very long protocols (owing to the low sample concentration the oxygen consumption in the O2k-Chamber is slower, therefore, it takes longer to reach the required oxygen concentrations.)
- - Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate.
- - Reoxygenation and nitrogen injection could lead to bubble formation in the O2k-Chamber.
- - CIV activity and cytochrome c test cannot be performed together with the fluorescence.
Compare SUIT protocols
- SUIT-018 AmR mt D041a simple protocol for simultaneous determination of O2 and H2O2 flux in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) changing the oxygen concentration in the same protocol.
- SUIT-018 AmR ce-pce D068 a protocol for simultaneous determination of O2 and H2O2 flux in permeabilized cells.
- SUIT-006 AmR mt D048 to investigate the dependence of H2O2 flux on mt-membrane potential on the N-control state in mitochondrial preparations.
- SUIT-009 to investigate H2O2 production driven by the reverse electron transfer (RET) in isolated mitochondria, tissue homogenate and permeabilized cells.
References
Year | Reference | Organism | Tissue;cell | |
---|---|---|---|---|
MiPNet24.10 H2O2 flux analysis | 2021-10-22 | Hydrogen peroxide flux analysis using Amplex UltraRed assay in MiR05-Kit with DatLab 7.4 | ||
Komlodi 2021 BEC AmR-O2 | 2021 | KomlΓ³di T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. Bioenerg Commun 2021.4. https://doi.org/10.26124/bec:2021-0004 | Saccharomyces cerevisiae | Other cell lines |
MiPNet20.14 AmplexRed H2O2-production | 2019-06-24 | O2k-FluoRespirometry: HRR and simultaneous determination of H2O2 production with Amplex UltraRed. | Mouse | Heart |
Komlodi 2018 Methods Mol Biol | 2018 | Komlodi T, Sobotka O, Krumschnabel G, Bezuidenhout N, Hiller E, Doerrier C, Gnaiger E (2018) Comparison of mitochondrial incubation media for measurement of respiration and hydrogen peroxide production. Methods Mol Biol 1782:137-55. | Human Mouse | Skeletal muscle HEK |
Makrecka-Kuka 2015 Biomolecules | 2015 | Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. https://doi.org/10.3390/biom5031319 | Human Mouse | Heart Nervous system HEK |
Krumschnabel 2015 Methods Mol Biol | 2015 | Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61. | Mouse | Nervous system |