SUIT-009 O2 ce-pce D016: Difference between revisions
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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 | ||
Originially 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. ย | |||
For O2-Application, choose [[SUIT-009 O2 mt D015]]; for O2 and H<sub>2</sub>O<sub>2</sub> measurements (AmR), choose: | 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__ | ||
ย Communicated by [[Iglesias-Gonzalez J]],ย [[Komlodi T]] and [[Gnaiger E]] (last update 2019-01-23) | ย Communicated by [[Iglesias-Gonzalez J]],ย [[Komlodi T]] and [[Gnaiger E]] (last update 2019-01-23) | ||
== SUIT protocols == | == SUIT protocols == | ||
* [[SUIT-009 AmR mt D021]] simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux on isolated mitochondria and tissue homogenate.ย | |||
* [[SUIT-009 O2 mt D015]] determination of O<sub>2</sub> flux on isolated mitochondria and tissue homogenate as a control for SUIT-009 AmR mt D021. | |||
* [[SUIT-009 AmR pce D019]] simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux on permeabilized cells.ย | |||
* [[SUIT-009 O2 pce D016]] determination of O<sub>2</sub> flux on permeabilized cells as a control for SUIT-009 AmR pce D019.ย | |||
== References == | == References == |
Revision as of 11:16, 28 January 2019
Description
Abbreviation: NS(P)
Reference: A: SUIT-009 short protocol for mt-respiration in pce.
SUIT number: D016_ce1;1Dig;1S;2D;3P;4Rot;5Ama
MitoPedia concepts:
SUIT protocol,
SUIT B,
Find
MitoPedia methods:
Respirometry
- SUIT-category: NS(P)
- SUIT protocol pattern: orthogonal 1S;2D;3P;4Rot;5Ama
Originially 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 forSUIT-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 2019-01-23)
SUIT protocols
- SUIT-009 AmR mt D021 simultaneous determination of O2 and H2O2 flux on isolated mitochondria and tissue homogenate.
- SUIT-009 O2 mt D015 determination of O2 flux on isolated mitochondria and tissue homogenate as a control for SUIT-009 AmR mt D021.
- SUIT-009 AmR pce D019 simultaneous determination of O2 and H2O2 flux on permeabilized cells.
- SUIT-009 O2 pce D016 determination of O2 flux on permeabilized cells as a control for SUIT-009 AmR pce D019.
References
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
- 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
- For H2O2-Application, choose SUIT-009 AmR pce D019; for O2 and H2O2 measurements (AmR), 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.
- - In some cell lines e.g. HEK293T, P does not have any effect because of the downregulation of the N-linked pathway.