Difference between revisions of "SUIT-009 O2 ce-pce D016"
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::: '''[[Categories of SUIT protocols |SUIT-category]]:''' NS(P) | ::: '''[[Categories of SUIT protocols |SUIT-category]]:''' NS(P) | ||
::: '''[[SUIT protocol pattern]]:''' orthogonal 1S;2D;3P;4Rot;5Ama | ::: '''[[SUIT protocol pattern]]:''' orthogonal 1S;2D;3P;4Rot;5Ama | ||
This is a short protocol for simultaneous determination of O<sub>2</sub> flux and the rate of [[MiPNet20.14 AmplexRed H2O2-production|H<sub>2</sub>O<sub>2</sub> production]] (H<sub>2</sub>O<sub>2</sub> flux) on permeabilized cells. It is used as a control experiment for [[SUIT-009 AmR pce D019]] without using the Amplex UltraRed assay in order to detect the effect of the fluorescent dye on the O<sub>2</sub> flux. We do not recommend this protocol for determination of the O<sub>2</sub> flux in the absence of Rotenone. | This is a short protocol for simultaneous determination of O<sub>2</sub> flux and the rate of [[MiPNet20.14 AmplexRed H2O2-production|H<sub>2</sub>O<sub>2</sub> production]] (H<sub>2</sub>O<sub>2</sub> flux) on permeabilized cells. It is used as a control experiment for [[SUIT-009 AmR ce-pce D019]] without using the Amplex UltraRed assay in order to detect the effect of the fluorescent dye on the O<sub>2</sub> flux. We do not recommend this protocol for determination of the O<sub>2</sub> flux in the absence of Rotenone. | ||
For O2-Application with isolated mitochondria and tissue homogenate, choose [[SUIT-009 O2 mt D015]]; for O2 and H<sub>2</sub>O<sub>2</sub> measurements (AmR) with permeabilized cells, choose [[SUIT-009 AmR pce D019]]. | For O2-Application with isolated mitochondria and tissue homogenate, choose [[SUIT-009 O2 mt D015]]; for O2 and H<sub>2</sub>O<sub>2</sub> measurements (AmR) with permeabilized cells, choose [[SUIT-009 AmR ce-pce D019]]. | ||
__TOC__ | __TOC__ | ||
Communicated by [[Iglesias-Gonzalez J]], '''[[Komlodi T]]''' and [[Gnaiger E]] (last update 2019-02-12) | Communicated by [[Iglesias-Gonzalez J]], '''[[Komlodi T]]''' and [[Gnaiger E]] (last update 2019-02-12) | ||
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:::* We do not recommend this protocol for respiration, since rotenone is not added at the beginning to avoid oxaloacetate inhibition towards succinate dehydrogenase. This protocol can be used as a control to investigate the effect of the fluorescence dyes on the respiration. | :::* We do not recommend this protocol for respiration, since rotenone is not added at the beginning to avoid oxaloacetate inhibition towards succinate dehydrogenase. This protocol can be used as a control to investigate the effect of the fluorescence dyes on the respiration. | ||
:::* It is a protocol to analyse the S- and NS-pathway linked respiration. | :::* It is a protocol to analyse the S- and NS-pathway linked respiration. | ||
:::* For H<sub>2</sub>O<sub>2</sub>-Application, choose [[SUIT-009 AmR pce D019]]. | :::* For H<sub>2</sub>O<sub>2</sub>-Application, choose [[SUIT-009 AmR ce-pce D019]]. | ||
:::+ Cytochrome ''c'' test can be performed in the S-OXPHOS state. | :::+ Cytochrome ''c'' test can be performed in the S-OXPHOS state. | ||
:::+ Reasonable duration of the experiment. | :::+ Reasonable duration of the experiment. | ||
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::: [[SUIT-006 O2 mt D022]]: to investigate the S(Rot)-linked respiration. | ::: [[SUIT-006 O2 mt D022]]: to investigate the S(Rot)-linked respiration. | ||
== References == | == References == | ||
{{#ask:[[Category:Publications]] [[Additional label::SUIT-009 O2 pce D016]] [[Additional label::O2k-Protocol]] | {{#ask:[[Category:Publications]] [[Additional label::SUIT-009 O2 ce-pce D016]] [[Additional label::O2k-Protocol]] [[Instrument and method::O2k-Protocol]] | ||
| ?Was published in year=Year | | ?Was published in year=Year | ||
| ?Has title=Reference | | ?Has title=Reference | ||
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| offset=0 | | offset=0 | ||
| sort=Was published in year | | sort=Was published in year | ||
| order=descending | | order=descending | ||
}} | }} |
Revision as of 19:34, 27 March 2019
Description
Abbreviation: NS(P)
Reference: B: short protocol for mt-respiration in pce- SUIT-009
SUIT number: D016_ce1;1Dig;1S;2D;3P;4Rot;5Ama
- MitoPedia: SUIT
- SUIT-category: NS(P)
- SUIT protocol pattern: orthogonal 1S;2D;3P;4Rot;5Ama
This is a short protocol for simultaneous determination of O2 flux and the rate of H2O2 production (H2O2 flux) on permeabilized cells. It is used as a control experiment for SUIT-009 AmR ce-pce D019 without using the Amplex UltraRed assay in order to detect the effect of the fluorescent dye on the O2 flux. We do not recommend this protocol for determination of the O2 flux in the absence of Rotenone. For O2-Application with isolated mitochondria and tissue homogenate, choose SUIT-009 O2 mt D015; for O2 and H2O2 measurements (AmR) with permeabilized cells, choose SUIT-009 AmR ce-pce D019.
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) |
---|---|---|---|---|
ce1 | ROUTINE | ce1
| ||
1Dig | Dig | ce1;1Dig
|
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1S | SL(n) | S | CII | 1S
|
2D | SP | S | CII | 1S;2D
|
2c | ScP | S | CII | 1S;2D;2c
|
3P | NSP | NS | CI&II | 1S;2D;3P
|
4Rot | SP | S | CII | 1S;2D;3P;4Rot
|
5Ama | ROX | 1S;2D;3P;4Rot;5Ama
|
Step | Respiratory state | Pathway control | ET-Complex | Comment |
---|---|---|---|---|
## AsTm | AsTmE | CIV | CIV | |
## Azd | CHB |
- 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
- We do not recommend this protocol for respiration, since rotenone is not added at the beginning to avoid oxaloacetate inhibition towards succinate dehydrogenase. This protocol can be used as a control to investigate the effect of the fluorescence dyes on the respiration.
- It is a protocol to analyse the S- and NS-pathway linked respiration.
- For H2O2-Application, choose SUIT-009 AmR ce-pce D019.
- + Cytochrome c test can be performed in the S-OXPHOS state.
- + Reasonable duration of the experiment.
- + This protocol can be extended with the Complex IV module.
- - A succinate concentration > 10 mM might be required to reach saturating S-OXPHOS capacity.
- - In some cell lines e.g. HEK293T, P does not have any effect because of the downregulation of the N-linked pathway.
Compare SUIT protocols
- SUIT-006 O2 mt D022: to investigate the S(Rot)-linked respiration.
References
MitoPedia concepts: SUIT protocol, SUIT B, Find
MitoPedia methods:
Respirometry