Difference between revisions of "SUIT-003 O2 ce-pce D018"
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:::* [[CIV]] activity can also be measured after the cell viability test module. | :::* [[CIV]] activity can also be measured after the cell viability test module. | ||
:::+ 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. | :::+ 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. | ||
:::- The addition of [[oligomycin]] has to be carefully tested to avoid inhibitory effects in the evaluation of the [[ET | :::- 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. | :::- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler. | ||
Revision as of 15:50, 8 June 2020
Description
Abbreviation: CCVP-Glc
Reference: C SUIT-003 Second version of the Coupling-control protocol and respirometric cell viability test (CCV protocol)
SUIT number: D018_ce1;ce1P;ce2Omy;ce3U;ce3Glc;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4Azd
O2k-Application: O2
- SUIT protocol pattern: ce1;ce1P;ce2Omy;ce3U;ce3Glc;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4Azd
SUIT-003 O2 ce-pce D018 is designed to study coupling control and plasma membrane permeability of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE, LEAK respiration and Electron transfer pathway in the presence of glucose. 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. At Electron transfer pathway state, glucose is added to test the Crabtree effect on living cells. The viability index, VI, in the simple CCVP-Glc (SUIT-003 O2 ce-pce D018) may be underestimated if succinate uptake in the living cells proceeds through the dicarboxylate carrier of the plasma membrane. CCVP-Glc can be extended by the Complex IV module.
Communicated by Doerrier C, Garcia-Souza LF and Iglesias-Gonzalez J (last update 2019-06-06)
Steps and respiratory states
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
| ||
ce3Glc | E | ce1;ce1P;ce2Omy;ce3U;ce3Glc
| ||
ce4Rot | ROX | ce1;ce1P;ce2Omy;ce3U;ce3Glc;ce4Rot
| ||
ce5S | ce1;ce1P;ce2Omy;ce3U;ce3Glc;ce4Rot;ce5S
| |||
1Dig | SE | ce1;ce1P;ce2Omy;ce3U;ce3Glc;ce4Rot;ce5S;1Dig
| ||
1Ama | ROX | ce1;ce1P;ce2Omy;ce3U;ce3Glc;ce4Rot;ce5S;1Dig;2Ama
|
Step | Respiratory state | Pathway control | ET-Complex | Comment |
---|---|---|---|---|
## AsTm | AsTmE | CIV | CIV | |
## Azd | CHB |
- 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
- The SUIT-003 O2 ce-pce D018 is a cell coupling-control protocol. The total cell count (Nce) is the sum of viable cells (Nvce) and dead cells (Ndce).
- When MiR05 is used, the addition of pyruvate is recommended to provide an external fuel substrate.
- CIV activity can also be measured after the cell viability test module.
- + 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.
- - 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-pce D020 (A Advantageous alternative protocol)
- SUIT-003 O2 ce-pce D013
References
MitoPedia concepts: SUIT protocol, SUIT C, Find
MitoPedia methods:
Respirometry