SUIT-003 O2 ce D061

high-resolution terminology - matching measurements at high-resolution

SUIT-003 O2 ce D061

Description

Abbreviation: ce

Reference: B - complex cell coupling control protocol (CCP) with cell permeable compound NV161-SUIT-003

SUIT protocol pattern: ce1;ce2Omy;ce3U;ce4Rot;ce5S;ce6Mnanv;ce7Ama

SUIT-003 O2 ce-pce D061 is designed to study coupling control and the presence of succinate transporters in the plasma membrane of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE, LEAK and ET. To study LEAK-respiration, the phosphorylation system is inhibited by oligomycin. The final concentration of oligomycin has to be carefully optimized for various cell types, to minimize the inhibitory effect on the electron transfer system which would lead to an underestimation of ET-capacity. The addition of succinate after rotenone will allow us to stimulate the respiration in living cells expressing a succinate transporter in the plasma membrane and those cells that present a damage plasma membrane and are permeable to the substrate. However, the use of Mnanv allows us to differentiate between these two possible sources of stimulation in the respiration due that the permeable compounds only work in living cells which plasma membrane has not been altered due that to be active required to be cleavage by esterases present in the cytosol. The living cells with an altered permeability of the plasma membrane will not be affected by the used of Mnanv.

Communicated by  Garcia-Souza LF and Gnaiger E(last update 2019-06-24)

Steps and respiratory states

Step	State	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(Omy)	ce1;(ce2Omy) Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin. The addition of oligomycin is optional depending on the experimental question to resolve.
ce3U	E	cce1;(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.
ce5DMSO	ROX	ce1;(ce2Omy);ce3U;ce4Rot;ce5DMSO 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.
ce6DMSO	ROX	ce1;(ce2Omy);ce3U;ce4Rot;ce5DMSO;ce6DMSO 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.
ce7Ama	ROX	ce1;(ce2Omy);ce3U;ce4Rot;ce5DMSO;ce6DMSO;ce7Ama 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

Work in progress

The SUIT-003 O2 ce D061 is a cell coupling control protocol. The total cell count (N_ce) is the sum of viable cells (N_vce) and dead cells (N_dce).
CIV activity can also be measured after the cell viability test module.

- The addition of oligomycin has to be carefully tested to avoid inhibitory effects in the evaluation of the ET-capacity.

- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.

Compare SUIT protocols

SUIT-003 O2 ce D060 CCP with cell permeable compounds NV118 and NV161
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;ce2U-
SUIT-003 Ce1;ce2SD;ce3Omy;ce4U-
SUIT-003 Ce1;ce2SD;ce3U;ce4Rot;ce5Ama
SUIT-003 Ce1;ce2U;ce3Rot;ce4S;ce5Ama
SUIT-006: Coupling control protocol for mt-preparations (mtprep), counterpart to SUIT-003 for non-permeabilized cells (ce).

References

MitoPedia concepts: SUIT protocol, SUIT B, Find

MitoPedia methods: Respirometry