SUIT-003 O2 ce-pce D020

high-resolution terminology - matching measurements at high-resolution

SUIT-003 O2 ce-pce D020

Description

Abbreviation: CCVP

Reference: A SUIT-003 - Coupling-control protocol and respirometric cell viability test; ce: living cells, pce: permeabilized cells

SUIT number: D020_ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c;2Ama;3AsTm;4Azd

O2k-Application: O2

SUIT-003 O2 ce-pce D020 is designed to study coupling control and plasma membrane permeability of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE, LEAK respiration and Electron transfer pathway. 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 viability index, VI, in the simple CCVP-ce (SUIT-003 O2 ce-pce D020) may be underestimated, if succinate uptake in the living cells proceeds throught the dicarboxylate carrier of the plasma membrane. CCVP-ce can be extended by the Complex IV module.

Communicated by Doerrier C, Cardoso LH, Gnaiger E (last update 2024-09-19)

Representative traces

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.
ce1P	ROUTINE	ce1;ce1P ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
ce2Omy	L(Omy)	ce1;ce1P;ce2Omy Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
ce3U	E	ce1;ce1P;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;ce1P;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.

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
ce5S				ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway. Succinate, S ( type S-pathway to Q).
1Dig*	S_E	S	CII	ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig Multiple Digitonin titrations for complete plasma membrane permeabilization. Noncoupled electron transfer state, ET state, with ET capacity E.
1c	S_E	S	CII	ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c Addition of cytochrome c yields a test for integrity of the mtOM (cytochrome c control efficiency). Stimulation by added cytochrome c would indicate an injury of the mtOM and limitation of respiration in the preceding state without added c due to loss of cytochrome c. Typically, cytochrome c is added immediately after the earliest ADP-activation step (OXPHOS capacity P with saturating [ADP]). Noncoupled electron transfer state, ET state, with ET capacity E.
2Ama	ROX			ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;1Dig;1c;2Ama Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).

Step	Respiratory state	Pathway control	ET-Complex	Comment
## AsTm	AsTm_E	CIV	CIV
## Azd	CHB

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Coupling control

» Coupling control state

» ET capacity

» OXPHOS capacity

» LEAK respiration

Pathway control

» Electron transfer pathway

» Fatty acid oxidation pathway control state, F

» NADH electron transfer-pathway state, N

» Succinate pathway control state, S

» NS-pathway control state, NS

» Glycerophosphate pathway control state, Gp

» Complex IV single step, CIV

» Anaplerotic pathway control state

Main fuel substrates

» Glutamate, G

» Glycerophosphate, Gp

» Malate, M

» Octanoylcarnitine, Oct

» Pyruvate, P

» Succinate, S

Glossary

» List of SUIT states

» SUIT concept

Strengths and limitations

The SUIT-003 O2 ce-pce D020 is a coupling-control protocol for living cells. The total cell count (N_ce) is the sum of viable cells (N_vce) and dead cells (N_dce).
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 into the experiment, Complex III inhibitors can be used instead of rotenone to reach Rox.

+ 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 the 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 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.

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

Compare SUIT protocols

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
ce1P	Pyruvate (P)
ce2Omy	Oligomycin (Omy)
ce3U	Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)	Can be substituted for other uncoupler
ce4Rot	Rotenone (Rot)
ce5S	Succinate (S)
1Dig	Digitonin (Dig)
1c	Cytochrome c (c)
2Ama	Antimycin A (Ama)

Step	Chemical(s) and link(s)	Comments
## AsTm	Ascorbate (As) and TMPD (Tm)
## Azd	Azide (Azd)

Suggested stock concentrations are shown in the specific DL-Protocol.

References

	Year	Reference	Organism	Tissue;cell
Doerrier 2018 Methods Mol Biol	2018	Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. https://doi.org/10.1007/978-1-4939-7831-1_3	Human Mouse Rat Saccharomyces cerevisiae	Heart Skeletal muscle Endothelial;epithelial;mesothelial cell Blood cells HEK Platelet
Stadlmann 2006 Cell Biochem Biophys	2006	Stadlmann S, Renner K, Pollheimer J, Moser PL, Zeimet AG, Offner FA, Gnaiger E (2006) Preserved coupling of oxidative phosphorylation but decreased mitochondrial respiratory capacity in IL-1ß treated human peritoneal mesothelial cells. Cell Biochem Biophys 44:179-86.	Human	Endothelial;epithelial;mesothelial cell

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Respirometry