SUIT-009 O2 ce-pce D016: Difference between revisions
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{{MitoPedia | {{MitoPedia | ||
|abbr=NS(P) | |abbr=NS(P) | ||
|description=[[File: | |description=[[File:Ce1;1Dig;1S;2D;3P;4Rot;5Ama.png|500px|SUIT-009]] [[File:SUIT-009 O2 pce DL-File.png|600px|SUIT9]] | ||
|info='''B''': '''[[SUIT-009]]''' short protocol for mt-respirationΒ in [[permeabilized cells |pce]]. | |info='''B''': '''[[SUIT-009]]''' short protocol for mt-respirationΒ in [[permeabilized cells |pce]]. | ||
|SUIT number=D016_ce1;1Dig;1S;2D;3P;4Rot;5Ama | |SUIT number=D016_ce1;1Dig;1S;2D;3P;4Rot;5Ama | ||
|O2k-Application=O2 | |O2k-Application=O2 | ||
}} | }} | ||
::: '''[[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 fluorescence 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. Β | |||
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 pce D019]]. | ||
__TOC__ | __TOC__ | ||
Β Communicated by [[Iglesias-Gonzalez J]],Β '''[[Komlodi T]]''' and [[Gnaiger E]] (last update 2019-02-12) | Β Communicated by [[Iglesias-Gonzalez J]],Β '''[[Komlodi T]]''' and [[Gnaiger E]] (last update 2019-02-12) | ||
{{Template:SUIT-009 O2 pce D016}} | |||
== Strengths and limitations == | |||
:::* It is a protocol to investigate the S- and NS-pathway linked respiration. | |||
:::* For H<sub>2</sub>O<sub>2</sub>-Application, choose [[SUIT-009 AmR 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 == | |||
== References == | == References == | ||
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| order=descending | | order=descending | ||
}} | }} | ||
::: [[SUIT-006 O2 mt D022]]: to investigate the S(Rot)-linked respiration. | ::: [[SUIT-006 O2 mt D022]]: to investigate the S(Rot)-linked respiration. | ||
{{MitoPedia concepts | {{MitoPedia concepts | ||
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|mitopedia method=Respirometry | |mitopedia method=Respirometry | ||
}} | }} | ||
{{MitoPedia O2k and high-resolution respirometry}} | |||
{{MitoPedia topics}} |
Revision as of 16:11, 14 February 2019
Description
Abbreviation: NS(P)
Reference: B: SUIT-009 short protocol for mt-respiration in pce.
SUIT number: D016_ce1;1Dig;1S;2D;3P;4Rot;5Ama
- 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 pce D019 without using the Amplex UltraRed assay in order to detect the effect of the fluorescence 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 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
- Β» Electron transfer pathway
- Β» Fatty acid oxidation pathway control state, F
- Β» NADH electron transfer-pathway state, N
- Β» Succinate pathway control state, S
- Β» NS-pathway control state, NS
- Β» Glycerophosphate pathway control state, Gp
- Β» Complex IV single step, CIV
- Β» Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- Β» Glutamate, G
- Β» Glycerophosphate, Gp
- Β» Malate, M
- Β» Octanoylcarnitine, Oct
- Β» Pyruvate, P
- Β» Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
- It is a protocol to investigate the S- and NS-pathway linked respiration.
- For H2O2-Application, choose SUIT-009 AmR 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
References
Year | Reference | Organism | Tissue;cell | |
---|---|---|---|---|
Chang 2018 Life Sciences Meeting 2018 Innsbruck AT | 2018 | pH dependence of mitochondrial respiration and H2O2 production in oral cancer cells β a pilot study. | Human | HEK Kidney |
- SUIT-006 O2 mt D022: to investigate the S(Rot)-linked respiration.
MitoPedia concepts: SUIT protocol, SUIT B, Find
MitoPedia methods:
Respirometry