SUIT-003: Difference between revisions
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|abbr=ce | |abbr=ce | ||
|description=[[File:Ce1; | |description=[[File:Ce1;ce2Omy;ce3U-.png|300px]] [[File:Ce5S;1Dig;1c-.png|350px]] | ||
|info='''A: Coupling control protocol, [[Intact cells |ce]]''' [[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/6/64/SUIT-003.pdf|Bioblast pdf]] ยป[http://wiki.oroboros.at/index.php/File:SUIT-003.pdf Versions] | |info='''A: Coupling control protocol, [[Intact cells |ce]]''' [[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/6/64/SUIT-003.pdf|Bioblast pdf]] ยป[http://wiki.oroboros.at/index.php/File:SUIT-003.pdf Versions] | ||
}} | }} |
Revision as of 12:59, 21 January 2019
Description
Abbreviation: ce
Reference: A: Coupling control protocol, ce ยปVersions
- SUIT-category: ce
- SUIT protocol pattern: linear cell (ce) coupling control protocol 1ce;2ceOmy;3ceU, or complex cell coupling control protocol ce1;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c;2Ama
DLP applications
- SUIT-003 O2 ce D009 for non-permeabilized cells with oligomycin
- SUIT-003_O2_ce_D012 for non-permeabilized cells with pyruvate and oligomycin
- SUIT-003 Ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama D000# for cell membrane permeability test CCP(S)
- SUIT-003 O2 ce-pce D020 coupling control protocol and respirometric cell viability test (CCV protocol)
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. | ||
ce2Omy | L | ce1;ce2Omy
Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
| ||
ce3U | E | ce1;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;ce2Omy;ce3U;ce4Rot
Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration. |
Strengths and limitations
- The SUIT-003 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.
- For determination of respirometric viability index, it is assumed that both viable and dead cells contain functional mitochondria.
- 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.
- + Respirometric cell viability test correlates with other cell viability quantification methods.
- + 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 addition of cytochrome c.
- + CIV activity can also be measured after the cell viability test 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).
- - Cells with succinate transporter are not suitable for the viability test.
- - Pathological profiles could hinder the cell viability test.
- - Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
Compare SUIT protocols
- 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;ce2P;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
MitoPedia concepts:
MiP concept,
SUIT protocol,
Recommended
MitoPedia methods:
Respirometry