Description
Abbreviation: NS(GM)
Reference: A: SUIT-018 for simultaneous determination of O2 and H2O2 flux on isolated mitochondria and tissue homogenate at low oxygen concentration (tissue normoxia). Β»Versions
SUIT number: D031_1GMS;2D;3Ama
O2k-Application: AmR
- SUIT-category: NS(GM)
- SUIT protocol pattern: linear 1GMS;2D;3Ama
SUIT-018 AmR mt D031 is a short protocol to study simultaneously the oxygen dependence of O2 flux and H2O2 production on isolated mitochondria. Is it possible to run the protocol under different oxygen concentration conditions: 1.) under tissue normoxia (50-60 Β΅M = [O2]), 2.) under tissue hyperoxia (> 120 Β΅M = [O2]) or 3.) you can change the oxygen concentration during the experiment. The oxygen concentration in the chamber can be altered injecting nitrogen with a syringe. We recommend to run SUIT-018 AmR mt D040 simultaneously at high oxygen concentration as a control.
Communicated by Iglesias-Gonzalez J, Komlodi T and Gnaiger E (last update 2019-02-12)
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
- SUIT-018 AmR mt D040 should be runned in parallel (tissue hyperoxia condition).
- + Reasonable duration of the experiment.
- + Simple protocol to evaluate the oxygen dependence of H2O2 production in LEAK and OXPHOS.
- - 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 D041 for simultaneous determination of O2 and H2O2 flux on isolated mitochondria and tissue homogenate changing the oxygen concentration in the same protocol.
- SUIT-006 AmR mt D048 to investigate the dependence of H2O2 flux on the mt-membrane potential on the N-control state in isolated mitochondria and tissue homogenate.
- SUIT-009 to investigate H2O2 production driven by the reverse electron transfer (RET) on 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 |
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Fluorometry