Difference between revisions of "SUIT-003 O2 ce D012"
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== Strengths and limitations == | == Strengths and limitations == | ||
:::* The SUIT-003 O2 ce D012 is a cell coupling control protocol. The total cell count (''N''<sub>ce</sub>) is the sum of viable cells (''N''<sub>vce</sub>) and dead cells (''N''<sub>dce</sub>). | :::* The [[SUIT-003 O2 ce D012]] is a cell [[coupling control protocol]]. The total cell count (''N''<sub>ce</sub>) is the sum of viable cells (''N''<sub>vce</sub>) and dead cells (''N''<sub>dce</sub>). | ||
:::* This protocol can be extended with the cell viability test module (See CCV [[SUIT-003 O2 ce-pce D020]]). | :::* This protocol can be extended with the cell viability test module (See CCV [[SUIT-003 O2 ce-pce D020]]). | ||
:::* When the cell viability test module is not incorporated to the experiment, complex III inhibitors can be used to reach Rox instead of rotenone. | :::* When the cell viability test module is not incorporated to the experiment, [[complex III]] inhibitors can be used to reach [[Residual oxygen consumption| Rox]] instead of [[rotenone]]. | ||
:::* When MiR05 is used, the addition of pyruvate is recommended to provide an external fuel substrate. | :::* 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. | :::* [[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. | ||
:::+ Flexibility of experiment duration: from 1 hour (cell coupling control protocol) to 2 and a half hours (cell coupling control, cell viability test module and CIV module). | :::+ Flexibility of experiment duration: from 1 hour (cell [[coupling control protocol]]) to 2 and a half hours (cell coupling control, cell viability test module and [[CIV]] module). | ||
:::- The addition of [[oligomycin]] has to be carefully tested to avoid inhibitory effects in the evaluation of the ET-capacity. | :::- 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 09:54, 22 January 2019
Description
Abbreviation: ceN(P)
Reference: A - SUIT-003 coupling control protocol (CCP) with pyruvate
SUIT number: D012_ce1;ce2P;ce3Omy;ce4U;ce5Rot;ce6Ama
O2k-Application: O2
- SUIT-category: ceN(P)
- SUIT protocol pattern: complex cell coupling control protocol ce1;ce2P;ce3Omy;ce4U;ce*
References
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
ce1 | ROUTINE | ce1
ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state. | ||
ce1P | ROUTINE | ce1;ce1P. Pyruvate, ROUTINE, R
ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state. | ||
ce2Omy | L | ce1;ce1P;ce2Omy
Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
| ||
ce3U | E | ce1;ce1P;ce2Omy;ce3U
Uncoupler titration (avoiding inhibition by high uncoupler concentrations) to obtain electron transfer (ET) capacity E (noncoupled ET-state). Test for limitation of OXPHOS capacity P by the phosphorylation system (ANT, ATP synthase, phosphate transporter) relative to ET capacity E in mt-preparations: E-P control efficiency and E-L coupling efficiency. In living cells: E-R control efficiency and E-L coupling efficiency. Noncoupled electron transfer state, ET state, with ET capacity E. | ||
ce4Rot | ROX | ce1;ce1P;ce2Omy;ce3U;ce4Rot
| ||
ce5Ama | ROX | ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5Ama
|
Strengths and limitations
- The SUIT-003 O2 ce D012 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).
- When the cell viability test module is not incorporated to the experiment, complex III inhibitors can be used to reach Rox instead of rotenone.
- 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.
- + Flexibility of experiment duration: from 1 hour (cell coupling control protocol) to 2 and a half hours (cell coupling control, cell viability test module and CIV module).
- - 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.
MitoPedia concepts:
SUIT protocol,
SUIT A,
Find
MitoPedia methods:
Respirometry