SUIT-003 O2 ce D009

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SUIT-003 O2 ce D009

Description

Ce1;ce2Omy;ce3U-.png

Abbreviation: ce

Reference: A: Coupling control in intact cells with oligomycin; ce: non-permeabilized cells -SUIT-003

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

O2k-Application: O2

MitoPedia: SUIT
SUIT-category: ce
SUIT protocol pattern: linear cell (ce) coupling control protocol ce1;ce2Omy;ce3U

SUIT-003 O2 ce D009 has been designed to study the coupling control state of intact cells. The different capacities are tested in the sequence OXPHOS, LEAK and ET. Also, in order to study the LEAK-respiration, we need to inhibit the phosphorylation system which in our protocol is achieved using oligomycin. However, the final concentration of oligomycin has to be carefully adjusted in preliminary experiments for different samples and, the inhibitory effect on the electron transfer system, has to be studied to avoid an underestimation of the ET-capacity.


Communicated by Iglesias-Gonzalez J, Krumschnabel G, Doerrier C, Cardoso LH and Gnaiger E (last update 2019-03-27)
MitoPedia: SUIT

Steps and respiratory states

Ce1;ce2Omy;ce3U-.png

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 SUIT-003 O2 ce D009 is a cell coupling control protocol. 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 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

References

 YearReferenceOrganismTissue;cell
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
Lymphocyte
MiPNet10.04 CellRespiration2016-08-08
O2k-Protocols
An experiment with high-resolution respirometry: coupling control in cell respiration.
MouseBlood cells
Gnaiger 2008 POS2008
O2k-Protocols contents
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.
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. Biochem J 380:919-28.HumanFibroblast
Ce1;ce2Omy;ce3U;ce4Rot;ce5Ama.png

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).
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 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