SUIT-018: Difference between revisions
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{{MitoPedia | {{MitoPedia | ||
|abbr=NS(GM) | |abbr=NS(GM) | ||
|description=[[File:1GMS;2D | |description=[[File:1GMS;2D-.png|300px|SUIT-018]] | ||
|info='''A: Short protocol for oxygen dependence of O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> production on isolated mitochondria or tissue homogenate''' [[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/6/64/SUIT-018.pdf|Bioblast pdf]]ยป[http://wiki.oroboros.at/index.php/File:SUIT-018.pdf Versions] | |info='''A: Short protocol for oxygen dependence of O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> production on isolated mitochondria or tissue homogenate''' [[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/6/64/SUIT-018.pdf|Bioblast pdf]]ยป[http://wiki.oroboros.at/index.php/File:SUIT-018.pdf Versions] | ||
}} | }} |
Revision as of 09:48, 29 May 2019
Description
Abbreviation: NS(GM)
Reference: A: Short protocol for oxygen dependence of O2 flux and H2O2 production on isolated mitochondria or tissue homogenate ยปVersions
- SUIT-category:NS(GM)
- SUIT protocol pattern: linear 1GMS;2D;-
SUIT-018 is a short protocol to study simultaneously the oxygen dependence of O2 flux and H2O2 production on isolated mitochondria or tissue homogenate (except of liver homogenate). Is it possible to run the protocol under different oxygen concentration conditions: 1.) under tissue normoxia (50-60 ยตM ~ [O2]) SUIT-018 AmR mt D031, 2.) under tissue hyperoxia (> ~120 ยตM = [O2]) SUIT-018 AmR mt D040 or 3.) modifying the oxygen concentration during the experiment SUIT-018 AmR mt D041. The oxygen concentration in the chamber can be altered injecting nitrogen with a syringe. It is recommended to run both SUIT-018 AmR mt D031 and SUIT-018 AmR mt D040 in parallel or SUIT-018 AmR mt D041 alone in order to test the H2O2 production at different oxygen concentrations.
Communicated by Komlodi T, Iglesias-Gonzalez J and Gnaiger E (last update 2019-04-03)
Specific SUIT protocols
- SUIT-018 AmR mt D031 simultaneous determination of O2 and H2O2 flux on isolated mitochondria, tissue homogenate and permeabilized cells at low oxygen concentration (tissue normoxia).
- SUIT-018 AmR mt D040 simultaneous determination of O2 and H2O2 flux on isolated mitochondria, tissue homogenate and permeabilized cells at high oxygen concentration (tissue hyperoxia).
- SUIT-018 AmR mt D041 simultaneous determination of O2 and H2O2 flux on isolated mitochondria, tissue homogenate and permeabilized cells changing the oxygen concentration in the same protocol.
- SUIT-018 O2 mt D054 to measure O2 flux on isolated mitochondria, tissue homogenate and permeabilized cells as a control to test the effect of the fluorescence dye on the respiration
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
- Many fluorescence dyes can inhibit components of the ET system and thus, affecting the respiration. Therefore, a control run of the protocol should be done in the absence of the fluorescence dye.
- + Short duration of the experiment.
- + Simple protocol to evaluate the oxygen dependence of H2O2 production in LEAK and OXPHOS state.
- - 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-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 | Komlรณdi 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: MiP concept, SUIT protocol, Recommended
MitoPedia methods:
Fluorometry