Description
Abbreviation: CCP ce-pce PM
Reference: A to analyse the coupling control on permeabilized cells in N(PM) pathway control state- SUIT-006
SUIT number: D029_ce1;1Dig;1PM;2D;2c;3Omy;4U;5Ama
O2k-Application: O2
- MitoPedia: SUIT
- SUIT protocol pattern: ce1;1Dig;1PM;2D;2c;3Omy;4U;5Ama
SUIT-006 O2 ce-pce D029 is a coupling-control protocol for permeabilized cells. Living cells are added in the chamber, allowing to analyse ROUTINE respiration. The cells are further permeabilized inside the O2k chamber, and different coupling control states are assessed (L(n) - P - L(Omy) - E) at N(PM) pathway control state. The use of oligomycin is optional, however, it provides important information when residual and endogenous adenylates are present (which may happen if ATPases are active in the sample). This situation may lead to overestimated LEAK respiration measured in the absence of adenylates - L(n). Therefore, oligomycin can be used to verify whether this occurs and obtain the LEAK state appropriately.
Communicated by Cardoso LH, Iglesias-Gonzalez J, Komlodi T and Doerrier C (last update 2019-06-06)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
ce1 | ROUTINE | ce1
| ||
1Dig | REN | ce1;1Dig
|
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M)
|
---|---|---|---|---|
1PM | PML(n) | N | CI | 1PM
|
2D | PMP | N | CI | 1PM;2D
|
2c | PMcP | N | CI | 1PM;2D;2c
|
3Omy | PMLOmy | N | CI | 1PM;2D;2c;3Omy
|
4U | PME | N | CI | 1PM;2D;2c;3Omy;4U
|
5Ama | ROX | 1PM;2D;2c;3Omy;4U;5Ama
|
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- 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
- This protocol is indicated when the aim is to analyse coupling control in permeabilized cells in NADH-pathway using PM as substrates.
- + Reasonable duration of the experiment.
- + This protocol can be extended with the Complex IV module.
- - This protocol analyses the coupling control in NADH-pathway (with PM), however, it is less specific than a similar protocol containing the complex II inhibitor malonic acid, that would allow to analyse the NADH-pathway without any contribution of the S-pathway.
- - This protocol does not include internal ET-pathway control steps.
- - Careful washing is required after the experiment to avoid carry-over of uncoupler and inhibitors, if used.
Compare SUIT protocols
- SUIT-001 O2 ce-pce D003: Extended protocol with SUIT-006 as the initial coupling control module, for permeabilized cells.
- SUIT-003: Coupling control protocol for non-permeabilized cells (ce), counterpart to SUIT-006 for mt-preparations (mtprep).
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Respirometry