SUIT-003: Difference between revisions
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{{MitoPedia | {{MitoPedia | ||
|abbr=ce | |abbr=CCP-ce | ||
|description=[[File:Ce1;ce2(Omy);ce3U-.png| | |description=[[File:Ce1;ce2(Omy);ce3U-.png|250px]] [[File:Ce5S;1Dig;1c-.png|250px]] | ||
|info='''A: Coupling control protocol, [[ | |info='''A: [[Coupling-control protocol]], [[living cells]]''' Β»[http://wiki.oroboros.at/images/d/da/MiPNet08.09_CellRespiration.pdf MiPNet08.09 CellRespiration Bioblast pdf] Β | ||
}} | }} | ||
::: '''[[SUIT protocol pattern]]:''' 1ce;2ceOmy;3ceU;ce*, or ce1;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c;2Ama | |||
::: '''[[SUIT protocol pattern]]:''' | Β | ||
The protocols recommended in the SUIT-003 have been designed to study the coupling control state of living cells. In order to study the [[LEAK respiration]], the phosphorylation system needs to be inhibited, which is achieved in this protocol using [[oligomycin]]. However, the [[oligomycin]] concentration has to be carefully adjusted for different samples and the inhibitory effect on the [[Electron transfer pathway|electron transfer system]] has to be studied to avoid an underestimation of the [[ET capacity]]. SUIT-003 follows a modular approach and can be easily used in combination with the permeability test and/or the assessment of the complex IV capacity. | |||
Β | |||
__TOC__ | __TOC__ | ||
Communicated by [[Cardoso LHD]], [[Doerrier C]], [[Huete-Ortega M]], [[Gnaiger E]] (last update 2021-03-23) | |||
== | == Specific SUIT protocols == | ||
Β | |||
=== SUIT-003 O2 ce D009 === | |||
[[File:1ce;2ceOmy;3ceU-.jpg|200px]] | [[File:1ce;2ceOmy;3ceU-.jpg|200px]] | ||
*Β [[SUIT-003 O2 ce D009]] for non-permeabilized cells with oligomycin | [[File:D009 Traces.png|405px]] | ||
[[File:ce1; | *Β [[SUIT-003 O2 ce D009]] for non-permeabilized cells with oligomycin. | ||
* [[SUIT-003 O2 ce-pce D020]] coupling control protocol and respirometric cell viability test (CCV protocol) | Β | ||
Β | |||
=== SUIT-003 O2 ce-pce D020 === | |||
[[File:ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4Azd.png|400px]] | |||
[[File:D020 O2 traces.png|405px]] | |||
* [[SUIT-003 O2 ce-pce D020]] coupling-control protocol and respirometric cell viability test (CCV protocol). | |||
Β | |||
Β | |||
=== SUIT-003 O2 ce D037 and SUIT-003 O2 ce D038 === | |||
[[File:Ce1;ce1Glc;ce2(Omy);ce3U;ce4Ama.png|300px]] | |||
[[File:D037 O2 traces.png|400px]] | |||
Β | |||
Β | |||
[[File:Ce1;ce2(Omy);ce3U;ce3Glc;ce3'U;ce4Ama.png|300px]] | |||
[[File:D038 O2 trace.png|405px]] | |||
* [[SUIT-003 O2 ce D037]] and [[SUIT-003 O2 ce D038]] for coupling-control protocol with Crabtree effect test. | |||
Β | |||
=== SUIT-003 O2 ce D039 === | |||
[[File:ce1;(ce2Omy);ce3U;ce4Rot;ce5Ama.jpg|200px]] | |||
[[File:D009 Traces_algae.png|405px]] | |||
*Β [[SUIT-003 O2 ce D039]] for non-permeabilized [[microalgal]] cells. | |||
=== SUIT-003 O2 ce D050 === | |||
[[File:ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama.png|300px]] | |||
[[File:D050_O2_traces.png|405px]] | |||
*Β [[SUIT-003 O2 ce D050]] to study CI-deficiency with the plasma membrane-permeable succinate ([[MitoKit-CII/Succinate-nv]]) in living cells. | |||
=== SUIT-003 pH ce D067 === | |||
[[File:SUIT-003 O2 ce D067 diagram.png|300px]] | |||
[[File:D067 O2 trace.png|400px]][[File:D067 pH trace.png|400px]] | |||
* [[SUIT-003 pH ce D067]] to study glycolysis with simultaneous measurement of respiration and proton flux, besides coupling control and Crabtree effect. | |||
{{Template:SUIT-003 ce1;ce2;ce3}} | {{Template:SUIT-003 ce1;ce2;ce3}} | ||
== Strengths and limitations == | == Strengths and limitations == | ||
:::* The SUIT-003 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 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. | :::* 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. | :::* 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 | :::* When the cell viability test module is not incorporated to the experiment, complex III inhibitors should be used to reach 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. | ||
:::+ Respirometric cell viability test correlates with other cell viability quantification methods. | :::+ 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. | :::+ With the extended module ([[SUIT-003 O2 ce-pce D020]]), 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. | :::+ 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). | :::+ 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. | :::+ Reasonable duration of the experiment. | ||
Β | |||
:::- Cells with a cell membrane succinate transporter are not suitable for the viability test. | |||
:::- Pathological profiles could hinder the cell 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. | :::- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler. | ||
== Compare SUIT protocols == | == Compare SUIT protocols == | ||
::::* [[ | ::::* [[SUIT-003 O2 ce D012]] for non-permeabilized cells with pyruvate and oligomycin | ||
::::* [[SUIT- | ::::* [[SUIT-003 O2 ce-pce D013]] first version of the Coupling-control protocol and respirometric cell viability test (CCV protocol) | ||
::::* [[ | ::::* [[SUIT-003 AmR ce D017]] this protocol has been designed to study the H<sub>2</sub>O<sub>2</sub> production in various coupling-control states and test the cell membrane permeability with the same protocol using living cells. | ||
::::* [[SUIT-003 O2 ce-pce D018]] second version of the Coupling-control protocol and respirometric cell viability test (CCV protocol) | |||
::::* [[SUIT-003 O2 ce D028]] for cell membrane permeability test CCP(S) | ::::* [[SUIT-003 O2 ce D028]] for cell membrane permeability test CCP(S) | ||
::::* [[SUIT-003 | ::::* [[SUIT-003 AmR ce D058]] for studying the effect of the components of the AmR assay on the respiration using intact cells. | ||
::::* [[SUIT-003 O2 ce | ::::* [[SUIT-003 AmR ce D059]] for studying the effect of the components of the AmR assay on the respiration using intact cells (control protocol). | ||
::::* [[SUIT-003 | ::::* [[SUIT-003 O2 ce D060]] coupling-control protocol with cell permeable compounds [[NV118/MitoKit-CII|NV118]] and [[NV161/MitoKit-CII|NV161]]. | ||
::::* [[SUIT-003 Ce1; | ::::* [[SUIT-003 O2 ce D061]] as a control of [[SUIT-003 O2 ce D060]] | ||
::::* [[SUIT-003 Ce1; | ::::* [[SUIT-003 O2 ce D062]] as a control of [[SUIT-003 O2 ce D050]]. | ||
::::* [[SUIT-003 Ce1; | ::::* [[SUIT-003 Ce1;ce3U-]] | ||
::::* [[SUIT-003 Ce1; | ::::* [[SUIT-003 Ce1;ce1SD;ce2Omy;ce3U-]] Β | ||
::::* [[SUIT-006]]: Coupling control protocol for mt-preparations (mtprep), counterpart to SUIT-003 for non-permeabilized cells (ce). | ::::* [[SUIT-003 Ce1;ce1SD;ce3U;ce4Rot;ce5Ama]] | ||
::::* [[SUIT-003 Ce1;ce3U;ce4Rot;ce5S;ce6Ama]] | |||
::::* [[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-006]]: Coupling-control protocol for mt-preparations (mtprep), counterpart to SUIT-003 for non-permeabilized cells (ce). | |||
== References == | |||
{{#ask:[[Category:Publications]] [[Instrument and method::O2k-Protocol]] [[Additional label::SUIT-003]] | |||
|?Was published in year=Year | |||
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|sort=Was published in year | |||
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{{MitoPedia concepts | {{MitoPedia concepts | ||
|mitopedia concept=MiP concept, SUIT protocol, Recommended | |mitopedia concept=MiP concept, SUIT protocol, Recommended | ||
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|mitopedia method=Respirometry | |mitopedia method=Respirometry | ||
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{{MitoPedia O2k and high-resolution respirometry}} | |||
{{MitoPedia topics}} |
Latest revision as of 13:11, 17 November 2023
Description
Abbreviation: CCP-ce
Reference: A: Coupling-control protocol, living cells Β»MiPNet08.09 CellRespiration Bioblast pdf
- SUIT protocol pattern: 1ce;2ceOmy;3ceU;ce*, or ce1;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c;2Ama
The protocols recommended in the SUIT-003 have been designed to study the coupling control state of living cells. In order to study the LEAK respiration, the phosphorylation system needs to be inhibited, which is achieved in this protocol using oligomycin. However, the oligomycin concentration has to be carefully adjusted for different samples and the inhibitory effect on the electron transfer system has to be studied to avoid an underestimation of the ET capacity. SUIT-003 follows a modular approach and can be easily used in combination with the permeability test and/or the assessment of the complex IV capacity.
Communicated by Cardoso LHD, Doerrier C, Huete-Ortega M, Gnaiger E (last update 2021-03-23)
Specific SUIT protocols
SUIT-003 O2 ce D009
- SUIT-003 O2 ce D009 for non-permeabilized cells with oligomycin.
SUIT-003 O2 ce-pce D020
- SUIT-003 O2 ce-pce D020 coupling-control protocol and respirometric cell viability test (CCV protocol).
SUIT-003 O2 ce D037 and SUIT-003 O2 ce D038
- SUIT-003 O2 ce D037 and SUIT-003 O2 ce D038 for coupling-control protocol with Crabtree effect test.
SUIT-003 O2 ce D039
- SUIT-003 O2 ce D039 for non-permeabilized microalgal cells.
SUIT-003 O2 ce D050
- SUIT-003 O2 ce D050 to study CI-deficiency with the plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) in living cells.
SUIT-003 pH ce D067
- SUIT-003 pH ce D067 to study glycolysis with simultaneous measurement of respiration and proton flux, besides coupling control and Crabtree effect.
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M)
|
---|---|---|---|---|
ce1 | ROUTINE | ce1
| ||
ce2(Omy) | L(Omy) | ce1;ce2Omy
| ||
ce3U | E | ce1;ce2Omy;ce3U
| ||
ce* | ROX | ce1;ce2Omy;ce3U;ce*
|
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Coupling control
- Pathway control
- Β» Electron transfer pathway
- Β» Fatty acid oxidation pathway control state, F
- Β» NADH electron transfer-pathway state, N
- Β» Succinate pathway control state, S
- Β» NS-pathway control state, NS
- Β» Glycerophosphate pathway control state, Gp
- Β» Complex IV single step, CIV
- Β» Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- Β» Glutamate, G
- Β» Glycerophosphate, Gp
- Β» Malate, M
- Β» Octanoylcarnitine, Oct
- Β» Pyruvate, P
- Β» Succinate, S
- Main fuel substrates
- Glossary
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 should 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 (SUIT-003 O2 ce-pce D020), 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).
- + Reasonable duration of the experiment.
- - Cells with a cell membrane 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 D012 for non-permeabilized cells with pyruvate and oligomycin
- SUIT-003 O2 ce-pce D013 first version of the Coupling-control protocol and respirometric cell viability test (CCV protocol)
- SUIT-003 AmR ce D017 this protocol has been designed to study the H2O2 production in various coupling-control states and test the cell membrane permeability with the same protocol using living cells.
- SUIT-003 O2 ce-pce D018 second version of the Coupling-control protocol and respirometric cell viability test (CCV protocol)
- SUIT-003 O2 ce D028 for cell membrane permeability test CCP(S)
- SUIT-003 AmR ce D058 for studying the effect of the components of the AmR assay on the respiration using intact cells.
- SUIT-003 AmR ce D059 for studying the effect of the components of the AmR assay on the respiration using intact cells (control protocol).
- SUIT-003 O2 ce D060 coupling-control protocol with cell permeable compounds NV118 and NV161.
- SUIT-003 O2 ce D061 as a control of SUIT-003 O2 ce D060
- SUIT-003 O2 ce D062 as a control of SUIT-003 O2 ce D050.
- SUIT-003 Ce1;ce3U-
- SUIT-003 Ce1;ce1SD;ce2Omy;ce3U-
- SUIT-003 Ce1;ce1SD;ce3U;ce4Rot;ce5Ama
- SUIT-003 Ce1;ce3U;ce4Rot;ce5S;ce6Ama
- 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-006: Coupling-control protocol for mt-preparations (mtprep), counterpart to SUIT-003 for non-permeabilized cells (ce).
References
Year | Reference | Organism | Tissue;cell | |
---|---|---|---|---|
MiPNet08.09 CellRespiration | 2016-08-11 | High-resolution respirometry and coupling-control protocol with living cells: ROUTINE, LEAK, ET-pathway, ROX. | Human | Blood cells Lymphocyte |
Gnaiger 2008 POS | 2008 | Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial dysfunction in drug-induced toxicity (Dykens JA, Will Y, eds) John Wiley & Sons, Inc, Hoboken, NJ:327-52. | Mouse | Blood cells |
Huetter 2004 Biochem J | 2004 | HΓΌtter E, Renner K, Pfister G, StΓΆckl P, Jansen-DΓΌrr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. https://doi.org/10.1042/BJ20040095 | Human | Fibroblast |
MitoPedia concepts: MiP concept, SUIT protocol, Recommended
MitoPedia methods:
Respirometry