SUIT-003 O2 ce D009

high-resolution terminology - matching measurements at high-resolution

SUIT-003 O2 ce D009

Description

Abbreviation: CCP-ce

Reference: A: - SUIT-003

SUIT number: D009_ce1;ce2Omy;ce3U;ce4Rot;ce5Ama

O2k-Application: O2

MitoPedia: SUIT - Coupling control protocol (CCP) in living cells (ce) with oligomycin (LEAK state)

SUIT protocol pattern: ce1;ce2Omy;ce3U

CCP-ce (SUIT-003 O2 ce D009) is designed to study coupling control 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.

Communicated by Iglesias-Gonzalez J, Krumschnabel G, Doerrier C, Cardoso LH, Gnaiger E (last update 2019-06-06)

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	LOmy			ce1;ce2Omy Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
ce3U	E			ce1;ce2Omy;ce3U Noncoupled electron transfer state, ET state, with ET capacity E.
ce*	ROX			ce1;ce2Omy;ce3U;ce* 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.

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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 CCP-ce (SUIT-003 O2 ce D009) is a coupling control protocol for living cells. In a living cell population, 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 cell viability test module.

+ Reasonable duration of the experiment.

- 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 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;ce2P;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, counterpart to SUIT-003 for non-permeabilized cells (ce).

References

	Year	Reference	Organism	Tissue;cell
MiPNet08.09 CellRespiration	2016-08-11	High-resolution respirometry and coupling-control protocol with living cells: ROUTINE, LEAK, ET-pathway, ROX.	Human	Blood cells Lymphocyte
Gnaiger 2008 POS	2008	Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial dysfunction in drug-induced toxicity (Dykens JA, Will Y, eds) John Wiley & Sons, Inc, Hoboken, NJ:327-52.	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. https://doi.org/10.1042/BJ20040095	Human	Fibroblast

ce1;ce2Omy;ce3U;ce4Rot;ce5Ama

• ce1;ce2Omy;ce3U;ce4Rot;ce5Ama

In this ceCCP, ROX is measured after stepwise titration of rotenone (ce4Rot) and antimycin A (ce5Ama). In many intact cells ROX measured after titration of rotenone (ce4Rot) is not or only slightly further inhibited by subsequent addition of antimycin A (ce5Ama).

Respiration in senescent human primary fibroblasts (0.2×10⁶ cells/ml). Arrows show steps in the titration regime of the coupling control protocol, inducing the following respiratory states.

• ce1: ROUTINE (R; routine state in cell-culture medium) in intact cells (ce);
• ce2Omy: LEAK (L; inhibition of ATP-synthase by 1 μg/ml oligomycin);
• ce3U: ET-state (E; maximal stimulation by uncoupling of oxidative phosphorylation in four subsequent titrations of the uncoupler, U, FCCP (2.5–4 μM final concentration);
• ce4Rot: ROX (rotenone, Rot; residual oxygen consumption).
• ce5Ama: ROX (antimycin A, Ama; residual oxygen consumption). Modified after Huetter_2004_Biochem J.

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry