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
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 LHD, 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)|
|Step||State||Pathway||Q-junction||Comment - Events (E) and Marks (M)
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- Pathway control
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.
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MitoPedia methods: Respirometry