SUIT-009 O2 ce-pce D016: Difference between revisions
m (Doerrier Carolina moved page SUIT-009 O2 pce D016 to SUIT-009 O2 ce-pce D016) |
No edit summary |
||
Line 2: | Line 2: | ||
|abbr=NS(P) | |abbr=NS(P) | ||
|description=[[File:Ce1;1Dig;1S;2D;2c;3P;4Rot;5Ama.png|510px|SUIT-009]] ย | |description=[[File:Ce1;1Dig;1S;2D;2c;3P;4Rot;5Ama.png|510px|SUIT-009]] ย | ||
|info='''B''': short protocol for mt-respiration | |info='''B''': short protocol for mt-respiration in ce: non-permeabilized cells, pce: permeabilized cells - '''[[SUIT-009]]''' ย | ||
|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 | ||
Line 9: | Line 9: | ||
::: '''[[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 | This is a short protocol for simultaneous determination of O<sub>2</sub> flux in [[Permeabilized cells| permeabilized cells]] (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol). It is recommended 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 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. ย | ||
ย | |||
__TOC__ | __TOC__ | ||
ย Communicated by [[Iglesias-Gonzalez J]], | ย Communicated by [[Iglesias-Gonzalez J]], [[Komlodi T]] and [[Gnaiger E]] (last update 2019-02-12) | ||
{{Template:SUIT-009 O2 pce D016}} | {{Template:SUIT-009 O2 pce D016}} | ||
== 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 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 ce-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. | ||
:::+ | :::+ Short duration of the experiment. | ||
:::+ This protocol can be extended with the Complex IV module. | :::+ This protocol can be extended with the Complex IV module. | ||
:::- A succinate concentration > 10 mM might be required to reach | :::- A succinate concentration > 10 mM might be required to reach S-OXPHOS capacity. ย | ||
:::- In some cell lines e.g. HEK293T, P does not have any effect because of the downregulation of the N-linked pathway. | :::- In some cell lines e.g. HEK293T, P does not have any effect because of the downregulation of the N-linked pathway. | ||
Revision as of 11:31, 28 March 2019
Description
Abbreviation: NS(P)
Reference: B: short protocol for mt-respiration in ce: non-permeabilized cells, pce: permeabilized cells - 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 in permeabilized cells (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol). It is recommended 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 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 2019-02-12)
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
- 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 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 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.
- + Short duration of the experiment.
- + This protocol can be extended with the Complex IV module.
- - A succinate concentration > 10 mM might be required to reach 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