SUIT-003

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SUIT-003

Description

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

Abbreviation: ce

Reference: A: Coupling control protocol, living cells »MiPNet08.09 CellRespiration Bioblast pdf

SUIT-category: ce
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 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 Iglesias-Gonzalez J, Cardoso LH, Doerrier C, Huete-Ortega M and Gnaiger E (last update 2019-03-28)

Specific SUIT protocols

SUIT-003 O2 ce D009

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

SUIT-003 O2 ce D012

Ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5Ama.png D012 O2 traces.png

SUIT-003 O2 ce-pce D018

Ce1;ce1P;ce2Omy;ce3U;ce4Glc;ce5Rot;ce6S;1Dig;1U;1c;2Ama;3AsTm;4Azd.png D018 O2 traces.png

  • 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

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

SUIT-003 O2 ce D028

Ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama.png D028 O2 traces.png

SUIT-003 O2 ce D037

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

SUIT-003 O2 ce D038

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 AmR ce D058

Ce1;ce2Omy;ce3U-.png D058 O2 traces merged.png D058 O2 traces AmR.png D058 O2 traces control.png

  • SUIT-003 AmR ce D058 for studying the effect of the components of the AmR assay on the respiration using intact cells
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) LOmy 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
MiPNet10.04 CellRespiration2016-08-08
O2k-Protocols
An experiment with high-resolution respirometry: coupling control in cell respiration.
MouseBlood cells
Gnaiger 2008 POS2008
O2k-Protocols contents
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.
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. Biochem J 380:919-28.HumanFibroblast

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry