SUIT-003 O2 ce D061

high-resolution terminology - matching measurements at high-resolution

SUIT-003 O2 ce D061

Description

Abbreviation: Carrier control of the plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) and plasma membrane-permeable malonate (MitoKit-CII/Malonate-nv) - SUIT-003 O2 ce D060

Reference: B

SUIT number: D061_ce1;(ce2Omy);ce3U;ce4Rot;ce5DMSO;ce6DMSO;ce7Ama

O2k-Application: O2

MitoPedia: SUIT - Coupling control protocol (CCP) with the carrier of the plasma membrane-permeable succinate ([MitoKit-CII/Succinate-nv]) and plasma membrane-permeable malonate ([MitoKit-CII/Malonate-nv]) in living cells (ce): DMSO.

SUIT protocol pattern: ce1;(ce2Omy);ce3U;ce4DMSO;ce5DMSO;ce6Rot;ce7Ama

SUIT-003 O2 ce D061 protocol is the control of the SUIT-003 O2 ce D060 adding the carrier DMSO in ROX instead of the plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) and plasma membrane-permeable malonate ([[MitoKit-CII/Malonate-nv]). Moreover, the SUIT-003 O2 ce D061 protocol allows to evaluate the coupling control of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE and ET. Optionally, to study LEAK-respiration, the phosphorylation system might be 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.

Communicated by  Garcia-Souza LF and Gnaiger E(last update 2019-06-27)

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(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

• The SUIT-003 O2 ce D060 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).
• This protocol can be extended with the cell viability test module (See CCV SUIT-003 O2 ce-pce D020).
• CIV activity can also be measured after the antimycin A titration or after the cell viability test module.

+ Reasonable duration of the experiment.

- To obtain LEAK respiration, the concentration 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 plasma membrane-permeable compounds MitoKit-CII/Succinate-nv and MitoKit-CII/Malonate-nv.
• 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