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.
- 1 Description
- 2 Specific SUIT protocols
- 3 Steps and respiratory states
- 4 Strengths and limitations
- 5 Compare SUIT protocols
- 6 References
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
- SUIT-003 O2 ce D009 for non-permeabilized cells with oligomycin
SUIT-003 O2 ce D012
- SUIT-003 O2 ce D012 coupling control protocol (CCP) with pyruvate
SUIT-003 O2 ce-pce D018
- 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 coupling control protocol and respirometric cell viability test (CCV protocol)
SUIT-003 O2 ce D028
- SUIT-003 O2 ce D028 for non-permeabilized cells with oligomycin
SUIT-003 O2 ce D037
- SUIT-003 O2 ce D037 coupling control protocol with Crabtree effect test
SUIT-003 O2 ce D038
- SUIT-003 O2 ce D038 coupling control protocol with Crabtree effect test
SUIT-003 O2 ce D039
SUIT-003 AmR ce D058
- SUIT-003 AmR ce D058 for studying the effect of the components of the AmR assay on the respiration using intact cells
Steps and respiratory states
|Step||State||Pathway||Q-junction|| Comment - Events (E) and Marks (M)
- » Selected keywords in SUIT protocols
- Pathway control
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 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;ce3U-
- SUIT-003 Ce1;ce1SD;ce2Omy;ce3U-
- SUIT-003 Ce1;ce1SD;ce3U;ce4Rot;ce5Ama
- 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 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.
|MiPNet08.09 CellRespiration||2016-08-11||Human||Blood cells|
|MiPNet10.04 CellRespiration||2016-08-08||Mouse||Blood cells|
|Gnaiger 2008 POS||2008||Mouse||Blood cells|
|Huetter 2004 Biochem J||2004||Hütter 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.||Human||Fibroblast|
MitoPedia methods: Respirometry