SUIT-003 O2 ce D012: Difference between revisions
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{{MitoPedia | {{MitoPedia | ||
|abbr= | |abbr=CCP-ce(P) | ||
|description=[[File: | |description=[[File:Ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5Ama.png|400px]] | ||
|info=''' | |info='''B''' - [[SUIT-003]] | ||
|application=O2 | |application=O2 | ||
}} | |SUIT number=D012_ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5Ama}} | ||
: | |||
{{Template:SUIT text D012}} | |||
__TOC__ | __TOC__ | ||
Communicated by [[Garcia-Souza LF]], [[Gnaiger E]] (last update 2019-06-24) | |||
{{Template:SUIT-003 O2 ce D012}} | {{Template:SUIT-003 O2 ce D012}} | ||
== 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>). | |||
:::* 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 [[Residual oxygen consumption| 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. | |||
== Compare SUIT protocols == | |||
::::* [[SUIT-003 O2 ce D028]] for cell membrane permeability test CCP(S) | |||
::::* [[SUIT-003 O2 ce-pce D013]] first version of the Coupling-control protocol and respirometric cell viability test (CCV protocol) | |||
::::* [[SUIT-003 O2 ce-pce D018]] second version of the Coupling-control protocol and respirometric cell viability test (CCV protocol) | |||
::::* [[SUIT-003 Ce1;ce1P;ce3U;ce4Glc;ce5M;ce6Rot;ce7S;1Dig;1c;2Ama;3AsTm;4Azd]] coupling-control protocol and respirometric cell viability test (CCV protocol) without oligomycin. | |||
::::* [[SUIT-003 Ce1;ce2U-]] | |||
::::* [[SUIT-003 Ce1;ce2SD;ce3Omy;ce4U-]] | |||
::::* [[SUIT-003 Ce1;ce2SD;ce3U;ce4Rot;ce5Ama]] | |||
::::* [[SUIT-003 Ce1;ce2U;ce3Rot;ce4S;ce5Ama]] | |||
::::* [[SUIT-006]]: Coupling-control protocol for mt-preparations (mtprep), counterpart to SUIT-003 for non-permeabilized cells (ce). | |||
== References == | == References == | ||
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{{MitoPedia concepts | |||
|mitopedia concept=SUIT protocol, SUIT B, Find | |||
}} | |||
{{MitoPedia methods | |||
|mitopedia method=Respirometry | |||
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Latest revision as of 23:51, 12 April 2021
Description
Abbreviation: CCP-ce(P)
Reference: B - SUIT-003
SUIT number: D012_ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5Ama
O2k-Application: O2
SUIT-003 O2 ce D012 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 pyruvate is recommended to support the cell respiration if the incubation media is not cell culture media. 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 known side effects over the ET-capacity. Rotenone and Antimycin A provide the ROX state and its sequential addition allows the extension of the protocol with the cell viability test module (See CCV SUIT-003 O2 ce-pce D020).
Communicated by Garcia-Souza LF, 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
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.
Compare SUIT protocols
- SUIT-003 O2 ce D028 for cell membrane permeability test CCP(S)
- SUIT-003 O2 ce-pce D013 first version of the Coupling-control protocol and respirometric cell viability test (CCV protocol)
- SUIT-003 O2 ce-pce D018 second version of the Coupling-control protocol and respirometric cell viability test (CCV protocol)
- SUIT-003 Ce1;ce1P;ce3U;ce4Glc;ce5M;ce6Rot;ce7S;1Dig;1c;2Ama;3AsTm;4Azd coupling-control protocol and respirometric cell viability test (CCV protocol) without oligomycin.
- SUIT-003 Ce1;ce2U-
- SUIT-003 Ce1;ce2SD;ce3Omy;ce4U-
- SUIT-003 Ce1;ce2SD;ce3U;ce4Rot;ce5Ama
- SUIT-003 Ce1;ce2U;ce3Rot;ce4S;ce5Ama
- SUIT-006: Coupling-control protocol for mt-preparations (mtprep), counterpart to SUIT-003 for non-permeabilized cells (ce).
References
MitoPedia concepts: SUIT protocol, SUIT B, Find
MitoPedia methods:
Respirometry