Difference between revisions of "SUIT-018 AmR mt D041"
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::: '''[[Categories of SUIT protocols |SUIT-category]]:''' NS(GM) | ::: '''[[Categories of SUIT protocols |SUIT-category]]:''' NS(GM) | ||
::: '''[[SUIT protocol pattern]]:''' orthogonal 1GMS;2D;3Ama | ::: '''[[SUIT protocol pattern]]:''' orthogonal 1GMS;2D;3Ama | ||
This is a short protocol to study simultaneously the oxygen dependence of O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> production on isolated mitochondria or tissue homogenate (except of liver homogenate). You can run the protocol 1.)under tissue normoxia (50-60 µM = [O<sub>2</sub>]) | This is a short protocol to study simultaneously the oxygen dependence of O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> production on isolated mitochondria or tissue homogenate (except of liver homogenate). You can run the protocol 1.)under tissue normoxia (50-60 µM = [O<sub>2</sub>]), 2.) under tissue hyperoxia [> 120 µM = [O<sub>2</sub>]] or 3.) you can change the oxygen concentration during the experiment. The oxygen concentration in the chamber can be changed by the injection of nitrogen with a syringe. | ||
__TOC__ | __TOC__ | ||
Communicated by [Komlodi T]] (last update 2019-02- | Communicated by '''[[Komlodi T]]''' (last update 2019-02-12) | ||
== References == | == References == | ||
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== Strengths and limitations == | == Strengths and limitations == | ||
:::+ Reasonable duration of the experiment. | :::+ Reasonable duration of the experiment. | ||
:::+ Simple protocol to evaluate the oxygen dependence of H<sub>2</sub>O<sub>2</sub> production. | :::+ Simple protocol to evaluate the oxygen dependence of H<sub>2</sub>O<sub>2</sub> production in LEAK and OXPHOS. | ||
:::- Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate. | :::- Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate. | ||
:::- H<sub>2</sub>O<sub>2</sub> production can be studied in two different states, LEAK and OXPHOS. | :::- H<sub>2</sub>O<sub>2</sub> production can be studied in two different states, LEAK and OXPHOS. |
Revision as of 13:01, 12 February 2019
Description
Abbreviation: NS(GM)
Reference: A: SUIT-018 to study simultaneously the oxygen dependence of O2 flux and H2O2 production in mitochondrial preparations (isolated mitochondria and tissue homogenate).
SUIT number: D041_1GMS;2D;3Ama
O2k-Application: AmR
- SUIT-category: NS(GM)
- SUIT protocol pattern: orthogonal 1GMS;2D;3Ama
This 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). You can run the protocol 1.)under tissue normoxia (50-60 µM = [O2]), 2.) under tissue hyperoxia [> 120 µM = [O2]] or 3.) you can change the oxygen concentration during the experiment. The oxygen concentration in the chamber can be changed by the injection of nitrogen with a syringe.
Communicated by Komlodi T (last update 2019-02-12)
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 | Komlódi 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 |
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
- + 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.
- - H2O2 production can be studied in two different states, LEAK and OXPHOS.
- - 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
MitoPedia concepts: SUIT protocol, SUIT A, Find
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