Abbreviation: CCP-ce Snv
Reference: A: - Coupling-control protocol for living cells with plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) - SUIT-003 - Plasma membrane-permeable succinate (NV118) in ROUTINE respiration. Coupling-control protocol (CCP) in living cells (ce)
SUIT number: D050_ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama
SUIT-003 O2 ce D050 protocol is designed to study CI-deficiency in living cells. Plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv, Snv), which is added in ROUTINE stimulates mitochondrial respiration in CI-deficient human blood cells, fibroblasts and heart fibres, acting on Complex II of the electron transfer system. Moreover, the SUIT-003 O2 ce D050 protocol enables evaluation of the coupling control of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE and Electron transfer pathway. Optionally, to study LEAK respiration, the phosphorylation system is inhibited by oligomycin. The final concentration of oligomycin has to be carefully optimized for various cell types, to minimize the inhibitory effect on the electron transfer system which would lead to an underestimation of ET capacity.
Communicated by Doerrier C (last update 2019-09-24)
Steps and respiratory states
|Step||State||Pathway||Q-junction||Comment - Events (E) and Marks (M)
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- Pathway control
Strengths and limitations
- + Reasonable duration of the experiment.
- + This is a cell coupling-control protocol that provides information on ROUTINE and Electron transfer pathway in the presence of (MitoKit-CII/Succinate-nv.
- - This protocol is provided without oligomycin addition. To also include LEAK respiration in the protocol, an event for the addition of oligomycin and a related mark can be added by editing the DLP file, creating a DLPU. However, the addition of oligomycin has to be carefully tested to avoid inhibitory effects in the evaluation of the ET capacity.
- - Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
Compare SUIT protocols
Chemicals and syringes
|Step||Chemical(s) and link(s)||Comments|
|(ce2Omy)||Oligomycin (Omy)||This step can be skipped|
|ce3U||Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)||Can be substituted for other uncoupler|
|ce5Ama||Antimycin A (Ama)|
- Suggested stock concentrations are shown in the specific DL-Protocol.
MitoPedia methods: Respirometry