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Difference between revisions of "SUIT-003"

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{{MitoPedia
{{MitoPedia
|abbr=ce
|abbr=CCP-ce
|description=[[File:Ce1;ce2(Omy);ce3U-.png|250px]] [[File:Ce5S;1Dig;1c-.png|250px]]
|description=[[File:Ce1;ce2(Omy);ce3U-.png|250px]] [[File:Ce5S;1Dig;1c-.png|250px]]
|info='''A: Coupling control protocol, [[living cells]]''' »[http://wiki.oroboros.at/images/d/da/MiPNet08.09_CellRespiration.pdf MiPNet08.09 CellRespiration Bioblast pdf]  
|info='''A: [[Coupling-control protocol]], [[living cells]]''' »[http://wiki.oroboros.at/images/d/da/MiPNet08.09_CellRespiration.pdf MiPNet08.09 CellRespiration Bioblast pdf]  
}}
}}
::: '''[[Categories of SUIT protocols |SUIT-category]]:''' ce
::: '''[[SUIT protocol pattern]]:''' 1ce;2ceOmy;3ceU;ce*, or ce1;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c;2Ama
::: '''[[SUIT protocol pattern]]:''' Mono- or bidirectional linear cell (ce) [[coupling control protocol]] 1ce;2ceOmy;3ceU;ce*, or complex cell [[coupling control protocol]] 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]], we need to inhibit the phosphorylation system which in our protocol is achieved 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.
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.


__TOC__
__TOC__
  Communicated by [[Iglesias-Gonzalez J]], [[Cardoso LH]], [[Doerrier C]], [[Huete-Ortega M]] and [[Gnaiger E]] (last update 2019-03-28)
  Communicated by [[Cardoso LHD]], [[Doerrier C]], [[Huete-Ortega M]], [[Gnaiger E]] (last update 2021-03-23)


== Specific SUIT protocols ==
== Specific SUIT protocols ==
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=== SUIT-003 O2 ce D009 ===
=== SUIT-003 O2 ce D009 ===
[[File:1ce;2ceOmy;3ceU-.jpg|200px]]
[[File:1ce;2ceOmy;3ceU-.jpg|200px]]
[[File:D009 Traces.png|400px]]
[[File:D009 Traces.png|405px]]
*  [[SUIT-003 O2 ce D009]] for non-permeabilized cells with oligomycin
*  [[SUIT-003 O2 ce D009]] for non-permeabilized cells with oligomycin.


=== SUIT-003 O2 ce D012 ===
[[File:Ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5Ama.png|300px]]
[[File:D012 O2 traces.png|400px]]
*  [[SUIT-003 O2 ce D012]] coupling control protocol (CCP) with pyruvate
=== SUIT-003 O2 ce-pce D018 ===
[[File:Ce1;ce1P;ce2Omy;ce3U;ce4Glc;ce5Rot;ce6S;1Dig;1U;1c;2Ama;3AsTm;4Azd.png|400px]]
[[File:D018 O2 traces.png|400px]]
* [[SUIT-003 O2 ce-pce D018]] coupling control protocol and respirometric cell viability test (CCV protocol) with Crabtree effect test


=== SUIT-003 O2 ce-pce D020 ===
=== SUIT-003 O2 ce-pce D020 ===
[[File:ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4Azd.png|400px]]  
[[File:ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4Azd.png|400px]]  
[[File:D020 O2 traces.png|400px]]
[[File:D020 O2 traces.png|405px]]
* [[SUIT-003 O2 ce-pce D020]] coupling control protocol and respirometric cell viability test (CCV protocol)
* [[SUIT-003 O2 ce-pce D020]] coupling-control protocol and respirometric cell viability test (CCV protocol).


=== SUIT-003 O2 ce D028 ===
[[File:ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama.png|200px]]
[[File:D028 O2 traces.png|400px]]
*  [[SUIT-003 O2 ce D028]] for non-permeabilized cells with oligomycin


=== SUIT-003 O2 ce D037 ===
=== SUIT-003 O2 ce D037 and SUIT-003 O2 ce D038 ===
[[File:Ce1;ce1Glc;ce2(Omy);ce3U;ce4Ama.png|300px]]
[[File:Ce1;ce1Glc;ce2(Omy);ce3U;ce4Ama.png|300px]]
[[File:D037 O2 traces.png|400px]]
[[File:D037 O2 traces.png|400px]]
* [[SUIT-003 O2 ce D037]] coupling control protocol with Crabtree effect test


=== SUIT-003 O2 ce D038 ===
 
[[File:Ce1;ce2(Omy);ce3U;ce3Glc;ce3'U;ce4Ama.png|300px]]
[[File:Ce1;ce2(Omy);ce3U;ce3Glc;ce3'U;ce4Ama.png|300px]]
[[File:D038 O2 trace.png|400px]]
[[File:D038 O2 trace.png|405px]]
* [[SUIT-003 O2 ce D038]] coupling control protocol with Crabtree effect test
* [[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 ===
[[File:ce1;(ce2Omy);ce3U;ce4Rot;ce5Ama.jpg|200px]]
[[File:ce1;(ce2Omy);ce3U;ce4Rot;ce5Ama.jpg|200px]]
[[File:D009 Traces_algae.png|400px]]
[[File:D009 Traces_algae.png|405px]]
*  [[SUIT-003 O2 ce D039]] for non-permeabilized [[microalgal]] cells  
*  [[SUIT-003 O2 ce D039]] for non-permeabilized [[microalgal]] cells.
 


=== SUIT-003 AmR ce D058 ===
=== SUIT-003 O2 ce D050 ===
* [[SUIT-003 AmR ce D058]] for studying the effect of the components of the AmR assay on the respiration using intact cells
[[File:ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama.png|300px]]
[[File:D050_O2_traces.png|405px]]
* [[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.
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:::* 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 ([[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.
:::+ 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).
:::+ Reasonable duration of the experiment.
:::+ Reasonable duration of the experiment.


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== Compare SUIT protocols ==
== Compare SUIT protocols ==
::::* [[File:ce1;ce1P;ce2Omy;ce3U-.png|200px]]
::::* [[SUIT-003 O2 ce D012]] for non-permeabilized cells with pyruvate and oligomycin
::::* [[SUIT-003 O2 ce D012]] for non-permeabilized cells with pyruvate and oligomycin
::::* [[File:ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama.png|250px]]
::::* [[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 O2 ce-pce D013]] first version of the Coupling control protocol and respirometric cell viability test (CCV protocol)
::::* [[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-pce D018]] second version of the Coupling control protocol and respirometric cell viability test (CCV protocol)
::::* [[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 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 O2 ce D060]] coupling-control protocol with cell permeable compounds [[NV118/MitoKit-CII|NV118]] and [[NV161/MitoKit-CII|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;ce3U-]]
::::* [[SUIT-003 Ce1;ce1SD;ce2Omy;ce3U-]]  
::::* [[SUIT-003 Ce1;ce1SD;ce2Omy;ce3U-]]  
::::* [[SUIT-003 Ce1;ce1SD;ce3U;ce4Rot;ce5Ama]]
::::* [[SUIT-003 Ce1;ce1SD;ce3U;ce4Rot;ce5Ama]]
::::* [[SUIT-003 Ce1;ce3U;ce4Rot;ce5S;ce6Ama]]
::::* [[SUIT-003 Ce1;ce3U;ce4Rot;ce5S;ce6Ama]]
::::* [[SUIT-006]]: Coupling control protocol for mt-preparations (mtprep), counterpart to SUIT-003 for non-permeabilized cells (ce).
::::* [[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 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-006]]: Coupling-control protocol for mt-preparations (mtprep), counterpart to SUIT-003 for non-permeabilized cells (ce).


== References ==
== References ==

Latest revision as of 12:11, 17 November 2023


high-resolution terminology - matching measurements at high-resolution


SUIT-003

Description

Ce1;ce2(Omy);ce3U-.png Ce5S;1Dig;1c-.png

Abbreviation: CCP-ce

Reference: A: Coupling-control protocol, living cells »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

Ce1;ce2Omy;ce3U-.png D009 Traces.png


SUIT-003 O2 ce-pce D020

Ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4Azd.png D020 O2 traces.png


SUIT-003 O2 ce D037 and SUIT-003 O2 ce D038

Ce1;ce1Glc;ce2(Omy);ce3U;ce4Ama.png D037 O2 traces.png


Ce1;ce2(Omy);ce3U;ce3Glc;ce3'U;ce4Ama.png D038 O2 trace.png


SUIT-003 O2 ce D039

Ce1;(ce2Omy);ce3U;ce4Rot;ce5Ama.jpg D009 Traces algae.png


SUIT-003 O2 ce D050

Ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama.png D050 O2 traces.png


SUIT-003 pH ce D067

SUIT-003 O2 ce D067 diagram.png D067 O2 trace.pngD067 pH trace.png

  • SUIT-003 pH ce D067 to study glycolysis with simultaneous measurement of respiration and proton flux, besides coupling control and Crabtree effect.
MitoPedia: SUIT

Steps and respiratory states

Ce1;ce2(Omy);ce3U-.png


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.
ce2(Omy) L(Omy) ce1;ce2Omy
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
  • The addition of oligomycin or its carrier (EtOH) is optional depending on the experimental question to resolve. If we add only the carrier we do not reach LEAK state and we will evaluate if the EtOH alters the ROUTINE state.
ce3U E ce1;ce2Omy;ce3U
ce* ROX ce1;ce2Omy;ce3U;ce*
  • 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.


Questions.jpg


Click to expand or collaps


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

References

 YearReferenceOrganismTissue;cell
MiPNet08.09 CellRespiration2016-08-11
O2k-Procedures contents
High-resolution respirometry and coupling-control protocol with living cells: ROUTINE, LEAK, ET-pathway, ROX.
HumanBlood cells
Lymphocyte
Gnaiger 2008 POS2008Gnaiger 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.MouseBlood cells
Huetter 2004 Biochem J2004Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. https://doi.org/10.1042/BJ20040095HumanFibroblast

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry