Description
Abbreviation: CCP-ce Snv
Reference: A: - Coupling control protocol for living cells with plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) - SUIT-003
SUIT number: D050_ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama
O2k-Application: O2
- MitoPedia: SUIT - Plasma membrane-permeable succinate (NV118) in ROUTINE respiration. Coupling control protocol (CCP) in living cells (ce)
- SUIT protocol pattern: 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 2019-06-26)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M)
|
---|---|---|---|---|
ce1 | ROUTINE | ce1
| ||
ce1Snv | ROUTINE | ce1
| ||
(ce2Omy) | L(Omy) | ce1;ce1Snv;(ce2Omy)
| ||
ce3U | E | ce1;ce1Snv;(ce2Omy);ce3U
| ||
ce4Rot | ROX | ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot
| ||
ce5Ama | ROX | ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama
|
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Coupling control
- Pathway control
- Main fuel substrates
- » Glutamate, G
- » Glycerophosphate, Gp
- » Malate, M
- » Octanoylcarnitine, Oct
- » Pyruvate, P
- » Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
- A carrier control with DMSO is recommended, SUIT-003 O2 ce D062 is provided with this purpose.
- + The use of (MitoKit-CII/Succinate-nv allows studying succinate-linked respiration, or to overcome a CI-deficiency in living cells.
- + Reasonable duration of the experiment.
- + This is a cell coupling control protocol that provides information on ROUTINE and ET in the presence of (MitoKit-CII/Succinate-nv.
- - This protocol is provided without oligomycin addition. To also include LEAK 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
- SUIT-003 O2 ce D062 carrier control SUIT protocol for SUIT-003 O2 ce D050 in living cells. The carrier of the plasma membrane-permeable succinate (DMSO) is added in ROUTINE.
- SUIT-003 O2 ce D060 CCP with cell permeable compounds (MitoKit-CII/Succinate-nv and MitoKit-CII/Malonate-nv).
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Respirometry