Difference between revisions of "SUIT-009 O2 mt D015"
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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 determination of O<sub>2</sub> flux in isolated mitochondria, tissue homogenate (except of liver) and permeabilized cells (already permeabilized when they are added to the chamber). It is | This is a short protocol for determination of O<sub>2</sub> flux in isolated mitochondria, tissue homogenate (except of liver) and permeabilized cells (already permeabilized when they are added to the chamber). It is recommended as a control experiment for [[SUIT-009 AmR mt D021]] without using the Amplex UltraRed assay in order to detect the effect of the fluorescent dye on the O<sub>2</sub> flux and with the cytochrome ''c'' to assess the integrity of the mitochondrial outer membrane. We do not recommend this protocol for determination of the O<sub>2</sub> flux in the absence of Rotenone. | ||
__TOC__ | __TOC__ | ||
Communicated by [[Iglesias-Gonzalez J]], [[Komlodi T]] and [[Gnaiger E]] (last update 2019-03-28) | Communicated by [[Iglesias-Gonzalez J]], [[Komlodi T]] and [[Gnaiger E]] (last update 2019-03-28) | ||
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== Strengths and limitations == | == Strengths and limitations == | ||
:::* We do not recommend this protocol | :::* We do not recommend this protocol to assess just respiration since rotenone is not added at the beginning and does not prevent the inhibitory effect of the oxaloacetate on the succinate dehydrogenase. This protocol should be used as a control to investigate the effect of the fluorescence dyes on the respiration. | ||
:::* It is a protocol to investigate the S- and NS-pathway linked respiration. | :::* It is a protocol to investigate the S- and NS-pathway linked respiration. | ||
:::* For H<sub>2</sub>O<sub>2</sub>-Application, | :::* For H<sub>2</sub>O<sub>2</sub>-Application, use [[SUIT-009 AmR mt D021]]. | ||
::: + Cytochrome ''c'' test can be performed in the S-OXPHOS state. | ::: + Cytochrome ''c'' test can be performed in the S-OXPHOS state. | ||
::: + | ::: + Short protocol. | ||
::: + This protocol can be extended with the Complex IV module. | ::: + This protocol can be extended with the Complex IV module. | ||
::: + Contribution of N pathway to the NS can be investigated after the addition of Rot. | ::: + Contribution of N-pathway to the NS can be investigated after the addition of Rot. | ||
::: - A succinate concentration > 10 mM might be required to reach | ::: - A succinate concentration > 10 mM might be required to reach S-OXPHOS capacity. | ||
::: - In some cell lines e.g. HEK293T, P | ::: - In some cell lines e.g. HEK293T, P has not any effect because of the downregulation of the N-linked pathway. | ||
== Compare SUIT protocols == | == Compare SUIT protocols == |
Revision as of 11:06, 28 March 2019
Description
Abbreviation: NS(P)
Reference: B: short protocol for respiration in mitochondrial preparations; mt: isolated mitochondria, tissue homogenate and permeabilized cells- SUIT-009
SUIT number: D015_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 determination of O2 flux in isolated mitochondria, tissue homogenate (except of liver) and permeabilized cells (already permeabilized when they are added to the chamber). It is recommended as a control experiment for SUIT-009 AmR mt D021 without using the Amplex UltraRed assay in order to detect the effect of the fluorescent dye on the O2 flux and with the cytochrome c to assess the integrity of the mitochondrial outer membrane. We do not recommend this protocol for determination of the O2 flux in the absence of Rotenone.
Communicated by Iglesias-Gonzalez J, Komlodi T and Gnaiger E (last update 2019-03-28)
Steps and respiratory states
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
|
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 to assess just respiration since rotenone is not added at the beginning and does not prevent the inhibitory effect of the oxaloacetate on the succinate dehydrogenase. This protocol should be used as a control to investigate the effect of the fluorescence dyes on the respiration.
- It is a protocol to investigate the S- and NS-pathway linked respiration.
- For H2O2-Application, use SUIT-009 AmR mt D021.
- + Cytochrome c test can be performed in the S-OXPHOS state.
- + Short protocol.
- + This protocol can be extended with the Complex IV module.
- + Contribution of N-pathway to the NS can be investigated after the addition of Rot.
- - A succinate concentration > 10 mM might be required to reach S-OXPHOS capacity.
- - In some cell lines e.g. HEK293T, P has not 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