Difference between revisions of "SUIT-006 AmR mt D048"
Line 2: | Line 2: | ||
|abbr=N(PM) | |abbr=N(PM) | ||
|description=[[File:1PM;2D;3Omy;4U;5Ama.png|400px]] | |description=[[File:1PM;2D;3Omy;4U;5Ama.png|400px]] | ||
|info='''A''': short protocol for simultaneous determination of O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> in mitochondrial preparations (isolated mitochondria | |info='''A''': short protocol for simultaneous determination of O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)- '''[[SUIT-006]]''' | ||
|application=AmR | |application=AmR | ||
|SUIT number=D048_1PM;2D;3Omy;4U;5Ama | |SUIT number=D048_1PM;2D;3Omy;4U;5Ama | ||
Line 9: | Line 9: | ||
::: '''[[Categories of SUIT protocols |SUIT-category]]:''' N(PM) | ::: '''[[Categories of SUIT protocols |SUIT-category]]:''' N(PM) | ||
::: '''[[SUIT protocol pattern]]:''' bidirectional linear [[coupling control protocol]] 1PM;2D;3Omy;4U;5Ama | ::: '''[[SUIT protocol pattern]]:''' bidirectional linear [[coupling control protocol]] 1PM;2D;3Omy;4U;5Ama | ||
This is a protocol to investigate the [[NADH Electron transfer-pathway state| N-]] control state in isolated mitochondria | This is a protocol to investigate the [[NADH Electron transfer-pathway state| N-]] control state in isolated mitochondria, tissue homogenate (except of liver homogenate) and permeabilized cells. Oligomycin (Omy) is used to induce a LEAK state of respiration via the inhibition of the ATP synthase. Since higher concentrations of Omy can decrease the ET state induced upon addition of uncoupler, the required concentration of Omy has to be determined. This is a short protocol to study simultaneously the O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> flux, in particular the dependence of H<sub>2</sub>O<sub>2</sub> flux on the mt-membrane potential. Addition of ADP depolarises the mt-membrane which leads to the decrease of the H<sub>2</sub>O<sub>2</sub> flux. [[Oligomycin|Omy]] hyperpolarises the mt-membrane resulting in increased H<sub>2</sub>O<sub>2</sub> production, while the addition of the uncoupler decreases the H<sub>2</sub>O<sub>2</sub> generation owing to the depolarisation of the mt-membrane. | ||
Line 21: | Line 21: | ||
:::* This protocol can be run under low oxygen concentration to test the oxygen dependence of mt-ROS production. For more technical details see: [[SUIT-018 AmR mt D031]], [[SUIT-018 AmR mt D041]]. | :::* This protocol can be run under low oxygen concentration to test the oxygen dependence of mt-ROS production. For more technical details see: [[SUIT-018 AmR mt D031]], [[SUIT-018 AmR mt D041]]. | ||
:::+ Reasonable duration of the experiment. | :::+ Reasonable duration of the experiment. | ||
:::- CIV activity cannot be determined and | :::- CIV activity cannot be determined and cytochrome ''c'' test cannot be performed together with the fluorescence dyes. | ||
:::+ This protocol can be extended with the Complex IV module in the following protocols: [[SUIT-006 O2 mt D047]]. | :::+ This protocol can be extended with the Complex IV module in the following protocols: [[SUIT-006 O2 mt D047]]. | ||
:::+ Cytochrome ''c'' test can be performed in the following protocols:[[SUIT-006 O2 mt D047]]. | :::+ Cytochrome ''c'' test can be performed in the following protocols:[[SUIT-006 O2 mt D047]]. |
Revision as of 11:22, 27 March 2019
Description
Abbreviation: N(PM)
Reference: A: short protocol for simultaneous determination of O2 flux and H2O2 in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)- SUIT-006
SUIT number: D048_1PM;2D;3Omy;4U;5Ama
O2k-Application: AmR
- MitoPedia: SUIT
- SUIT-category: N(PM)
- SUIT protocol pattern: bidirectional linear coupling control protocol 1PM;2D;3Omy;4U;5Ama
This is a protocol to investigate the N- control state in isolated mitochondria, tissue homogenate (except of liver homogenate) and permeabilized cells. Oligomycin (Omy) is used to induce a LEAK state of respiration via the inhibition of the ATP synthase. Since higher concentrations of Omy can decrease the ET state induced upon addition of uncoupler, the required concentration of Omy has to be determined. This is a short protocol to study simultaneously the O2 flux and H2O2 flux, in particular the dependence of H2O2 flux on the mt-membrane potential. Addition of ADP depolarises the mt-membrane which leads to the decrease of the H2O2 flux. Omy hyperpolarises the mt-membrane resulting in increased H2O2 production, while the addition of the uncoupler decreases the H2O2 generation owing to the depolarisation of the mt-membrane.
Communicated by Komlodi T (last update 2019-02-20)
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) |
---|---|---|---|---|
1PM | PML(n) | 1PM
| ||
2D | PMP | 1PM;2D
| ||
3Omy | PML(Omy) | 1PM;2D;3Omy
| ||
4U | PME | 1PM;2D;3Omy;4U
| ||
5Ama | ROX | 1PM;2D;3Omy;4U;5Ama
|
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Coupling control
- Pathway control
- Main fuel substrates
- » Glutamate, G
- » Glycerophosphate, Gp
- » Malate, M
- » Octanoylcarnitine, Oct
- » Pyruvate, P
- » Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
- This protocol can be run under low oxygen concentration to test the oxygen dependence of mt-ROS production. For more technical details see: SUIT-018 AmR mt D031, SUIT-018 AmR mt D041.
- + Reasonable duration of the experiment.
- - CIV activity cannot be determined and cytochrome c test cannot be performed together with the fluorescence dyes.
- + This protocol can be extended with the Complex IV module in the following protocols: SUIT-006 O2 mt D047.
- + Cytochrome c test can be performed in the following protocols:SUIT-006 O2 mt D047.
- - This protocol cannot be run with liver homogenate.
- - Omy concentration has to be determined. Higher concentrations of Omy may inhibit the ET state.
Compare SUIT protocols
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 |
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 |
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 |
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Fluorometry