SUIT-009 O2 mt D015: Difference between revisions
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::: '''[[SUIT protocol pattern]]:''' orthogonal 1S;2D;3P;4Rot;5Ama ย | ::: '''[[SUIT protocol pattern]]:''' orthogonal 1S;2D;3P;4Rot;5Ama ย |
Revision as of 15:38, 5 March 2019
Description
Abbreviation: NS(P)
Reference: B: SUIT-009 short protocol for respiration in mitochondrial preparations (isolated mitochondria and tissue homogenate).
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 simultaneous determination of O2 flux and the rate of H2O2 production (H2O2 flux) on isolated mitochondria, tissue homogenate (except of liver) and permeabilized cells. It is used as a control experiment forSUIT-009 AmR mt D021 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 permeabilized cells apply SUIT-009 O2 pce D016; for O2 and H2O2 measurements (AmR) with isolated mitochondria and tissue homogenate apply SUIT-009 AmR mt D021.
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) |
---|---|---|---|---|
1S | SL(n) | S | CII | 1S
|
2D | SP | 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 |
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- Coupling control
- Pathway control
- ยป Electron transfer pathway
- ยป Fatty acid oxidation pathway control state, F
- ยป NADH electron transfer-pathway state, N
- ยป Succinate pathway control state, S
- ยป NS-pathway control state, NS
- ยป Glycerophosphate pathway control state, Gp
- ยป Complex IV single step, CIV
- ยป Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- ยป Glutamate, G
- ยป Glycerophosphate, Gp
- ยป Malate, M
- ยป Octanoylcarnitine, Oct
- ยป Pyruvate, P
- ยป 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 investigate the S- and NS-pathway linked respiration.
- For H2O2-Application, choose SUIT-009 AmR mt D021.
- + 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.
- + 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 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