From Bioblast
Description
Abbreviation: ceS
Reference: B SUIT-003 coupling control protocol with succinate for permeability test
SUIT number: D028_ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama
O2k-Application: O2
- SUIT-category: ceS
- SUIT protocol pattern: linear cell coupling control protocol ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama
References
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
ce1 | ROUTINE | ce1
| ||
ce2Omy | L | ce1;ce2Omy
| ||
ce3U | E | ce1;ce2Omy;ce3U
| ||
ce4Rot | ROX | ce1;ce2Omy;ce3U;ce4Rot
|
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
ce5S | ce1;ce2Omy;ce3U;ce4Rot;ce5S
| |||
ce6Ama | ROX | ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama
|
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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
- The SUIT-003 Ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama is a cell coupling control protocol. The total cell count (Nce) is the sum of viable cells (Nvce) and dead cells (Ndce).
- This protocol can be extended with the cell viability test module (See CCV SUIT-003 O2 ce-pce D020).
- For determination of respirometric viability index, it is assumed that both viable and dead cells contain functional mitochondria.
- + With the extended module, S-ET capacity can be assessed after the performance of the coupling control protocol, as well as outer mt-membrane integrity with the addition of cytochrome c.
- + CIV activity can also be measured after the cell permeability test module.
- + Flexibility of experiment duration: from 1 hour (cell coupling control protocol) to 2 hours (permeability test module and CIV module).
- - Cells with a cell membrane succinate transporter are not suitable for the viability or permeability test.
- - 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.
ceR,L,E format
In the diagrams of coupling control protocols for intact cells (CCP), the steps may be replaced by the respiratory states.
- ce4Rot,ce5S;ce6Ama
- ce4Rot: In many intact cells ROX measured after titration of rotenone (ce4Rot) is not or only slightly further inhibited by subsequent titration of antimycin A.
- ce5S: Succinate is not permeable through the plasma membrane of many intact cells (Steinlechner-Maran 1997 Transplantation). If succinate is accessible to rotenone-inhibited uncoupled mitochondria in dead cells (dce), respiration will be stimulated.
- ce6Ama: Inhibition of CIII by antimycin A inhibits succinate-stimulated respiration (Stadlmann_2006_Cell Biochem Biophys).
MitoPedia concepts:
SUIT protocol,
SUIT B,
Find
MitoPedia methods:
Respirometry