SUIT-003 O2 ce D028: Difference between revisions
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|info='''B''' '''[[SUIT-003]]''' [[coupling control protocol]] with succinate for permeability test | |info='''B''' '''[[SUIT-003]]''' [[coupling control protocol]] with succinate for permeability test | ||
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::: '''[[SUIT protocol pattern]]:''' ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama | ::: '''[[SUIT protocol pattern]]:''' ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama | ||
SUIT-003 O2 ce D028 is a [[coupling control]] protocol for living cells. Different coupling control states are assessed (ROUTINE - L(Omy) - E) at ce-substrate state. The use of [[oligomycin]] provides important information about the ETS and TCA activity but the optimal concentration has to be carefully determined experimentally to avoid the well-known side effects over the ET-capacity. After the addition of [[Rotenone]] to reach ROX state we add succinate, which does not permeate the plasma membrane, to test if the plasma membrane of the cells is altered (permeability test). If we do not obtain further stimulation of the respiration we could conclude that the living cells are not permeable and remain intact. | |||
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ย Communicated by [[Doerrier C]], [[Iglesias-Gonzalez J]] and [[Gnaiger E]] (last update 2019-06- | ย Communicated by [[Doerrier C]], [[Iglesias-Gonzalez J]] and [[Gnaiger E]] (last update 2019-06-24) | ||
{{Template: SUIT-003 Ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama}} | {{Template: SUIT-003 Ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama}} |
Revision as of 22:22, 24 June 2019
Description
Abbreviation: S CCP permeability test
Reference: B SUIT-003 coupling control protocol with succinate for permeability test
- SUIT protocol pattern: ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama
SUIT-003 O2 ce D028 is a coupling control protocol for living cells. Different coupling control states are assessed (ROUTINE - L(Omy) - E) at ce-substrate state. The use of oligomycin provides important information about the ETS and TCA activity but the optimal concentration has to be carefully determined experimentally to avoid the well-known side effects over the ET-capacity. After the addition of Rotenone to reach ROX state we add succinate, which does not permeate the plasma membrane, to test if the plasma membrane of the cells is altered (permeability test). If we do not obtain further stimulation of the respiration we could conclude that the living cells are not permeable and remain intact.
Communicated by Doerrier C, Iglesias-Gonzalez J and Gnaiger E (last update 2019-06-24)
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
|
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Coupling control
- Pathway control
- ยป Electron transfer pathway
- ยป Fatty acid oxidation pathway control state, F
- ยป NADH electron transfer-pathway state, N
- ยป Succinate pathway control state, S
- ยป NS-pathway control state, NS
- ยป Glycerophosphate pathway control state, Gp
- ยป Complex IV single step, CIV
- ยป Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- ยป Glutamate, G
- ยป Glycerophosphate, Gp
- ยป Malate, M
- ยป Octanoylcarnitine, Oct
- ยป Pyruvate, P
- ยป 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).
References
MitoPedia concepts: SUIT protocol, SUIT B, Find
MitoPedia methods:
Respirometry