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SUIT-003 O2 ce D009

From Bioblast

high-resolution terminology - matching measurements at high-resolution

SUIT-003 O2 ce D009



Abbreviation: CCP-ce short

Reference: A: - SUIT-003 - Coupling-control protocol (CCP) in living cells (ce) with oligomycin (LEAK state)

SUIT number: D009_ce1;ce2Omy;ce3U;ce4Ama

O2k-Application: O2

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

Communicated by Doerrier C, Cardoso LHD, Gnaiger E (last update 2019-09-24)

Representative traces

D009 Traces.png

MitoPedia: SUIT

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
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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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 (Nce) is the sum of viable cells (Nvce) and dead cells (Ndce).
  • This protocol can be extended with the cell viability module. See CCV SUIT-003 O2 ce-pce D020.
  • CIV-activity can also be measured after application of the cell viability module.
+ Short 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

Chemicals and syringes

Step Chemical(s) and link(s) Comments
ce2Omy Oligomycin (Omy)
ce3U Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) Can be substituted for other uncoupler
ce4Ama Antimycin A (Ama)
Suggested stock concentrations are shown in the specific DL-Protocol.


MiPNet08.09 CellRespiration2016-08-11
O2k-Procedures contents
High-resolution respirometry and coupling-control protocol with living cells: ROUTINE, LEAK, ET-pathway, ROX.
HumanBlood cells
Gnaiger 2008 POS2008Gnaiger 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.MouseBlood cells
Huetter 2004 Biochem J2004Hütter 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.


  • ce1;ce2Omy;ce3U;ce4Rot;ce5Ama
In this ceCCP, ROX is measured after stepwise titration of rotenone (ce4Rot) and antimycin A (ce5Ama). In many living cells, Rox measured after titration of rotenone (ce4Rot) is not or only slightly further inhibited by subsequent addition of antimycin A (ce5Ama).
ceCCP (fibroblasts)

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 living 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