SUIT-003

high-resolution terminology - matching measurements at high-resolution

SUIT-003

Description

Abbreviation: ce

Reference: A  »Versions

SUIT-category: ce,S

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 D09
• SUIT-006_O2_ce_D12
• SUIT-7 O2 ce-pce D13
• SUIT-7 O2 ce-pce D18
• SUIT-7 O2 ce-pce D20
• Ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama
• Ce1;ce2P;ce3U;ce4Glc;ce5M;ce6Rot;ce7S;1Dig;1c;2Ama;3AsTm;4Azd
• Ce1;ce2SD;ce3Omy;ce4U-
• Ce1;ce2SD;ce3U;ce4Rot;ce5Ama
• Ce1;ce2U;ce3Rot;ce4S;ce5Ama
• SUIT-006

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. The addition of oligomycin or its carrier is optional depending on the experimental question to resolve.
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.
• This protocol can be extended with the cell viability test module.
• For determination of respirometric viability index, it is required cells with functional mitochondria from both intact and permeabilized (dead) cell population.
• When the cell viability test module is not incorporated to the experiment, complex III inhibitors can be used to reach Rox instead of rotenone.

+ 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

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 

MitoPedia methods: Respirometry