Description
Abbreviation: Has abbr::CCP(P) and cell membrane permeability test
Reference: [[Info::B - SUIT-003]]
SUIT number: Has SUIT number::D017_ce1;ce2P;ce3Omy;ce4U;ce5Rot;ce6S;ce7Ama
O2k-Application: Has application::O2
- MitoPedia: SUIT - coupling control protocol (CCP) with pyruvate and cell membrane permeability test
- SUIT protocol pattern: ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama
This protocol has been designed to study the H2O2 production in various coupling control states and test the cell membrane permeability with the same protocol using living cells. Pyruvate is added to support the ROUTINE respiration of living cells when they are in a respiratory for mitochondrial preparations (i.e. MiR05). To initiate LEAK-respiration oligomycin is added, inhibiting the ATP-synthase and leading to an increased in the H2O2 production. Whereas titrations of uncoupler results in a decreased H2O2 generation. However, one of the main problems with living cells is that the H2O2 flux is very low or hardly detectable due to the scavenger activity of the antioxidant enzymes in the cytosol.
Communicated by Komlodi T, Doerrier C, Iglesias-Gonzalez J and Cardoso LH (last update 2019-06-24)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
0DTPA |
| |||
0SOD |
| |||
0HRP |
| |||
0AmR |
|
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
ce1 | ROUTINE | ce1
| ||
ce1P | ROUTINE | ce1;ce1P; ROUTINE, R
| ||
ce2Omy | L(Omy) | ce1;ce1P;ce2Omy
| ||
ce3U | E | ce1;ce1P;ce2Omy;ce3U
| ||
ce4Rot | ROX | ce1;ce1P;ce2Omy;ce3U;ce4Rot
| ||
ce5S | ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S
| |||
ce6Ama | ROX | ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama
|
Strengths and limitations
- When MiR05 is used, the addition of pyruvate is recommended to provide an external fuel substrate.
- When culture medium (e.g. DMEM) is used, application of phenol red is not advisable, since it can interfere with the fluorescence signal.
- + Reasonable duration of the experiments.
- - Minor changes in the H2O2 flux can be seen with living cells because of the action of the antioxidant enzymes in the cytosol, which scavenge the H2O2 produced. (AmR is not permeable to the cell membrane, therefore, only the H2O2 that crosses the plasma membrane can be measured)
Compare SUIT protocols
- SUIT-013 AmR ce D023: SUIT-013 has been designed to study the oxygen dependence of H2O2 production in intact cells.
References
{{#ask: Additional label::SUIT-003 AmR ce D017 Instrument and method::O2k-Protocol | ?Was published in year=Year | ?Has title=Reference | ?Mammal and model=Organism | ?Tissue and cell=Tissue;cell | format=broadtable | limit=5000 | offset=0 | sort=Was published in year | order=descending }}
MitoPedia concepts:
SUIT protocol,
SUIT B,
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MitoPedia methods:
Fluorometry