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Difference between revisions of "SUIT-003 O2 ce D009"

From Bioblast
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|abbr=ce
|abbr=ce
|description=[[File:1ce;2ceOmy;3ceU-.jpg|150px]]
|description=[[File:1ce;2ceOmy;3ceU-.jpg|150px]]
|info='''A''' [[Huetter 2004 Biochem J]] - [[SUIT-003]]
|info='''A'''[[SUIT-003]]
|application=O2
|application=O2
|SUIT number=D009_ce1;ce2Omy;ce3U;ce4Rot;ce5Ama
|SUIT number=D009_ce1;ce2Omy;ce3U;ce4Rot;ce5Ama

Revision as of 15:36, 21 January 2019


high-resolution terminology - matching measurements at high-resolution


SUIT-003 O2 ce D009

Description

Ce1;ce2Omy;ce3U-.png

Abbreviation: ce

Reference: ASUIT-003

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

O2k-Application: O2

SUIT-category: ce
SUIT protocol pattern: linear cell (ce) coupling control protocol 1ce;2ceOmy;3ceU

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
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. https://doi.org/10.1042/BJ20040095HumanFibroblast
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.


Questions.jpg


Click to expand or collaps

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.
+ 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).
- The addition 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.

R;L;E format_ROX

R;L;E format ROX.jpg
  • The ceCCP diagram may show the respiratory states directly (left) as an alternative to presenting the titration steps.
  • Rot and Ama
In this ceCCP, ROX is measured after titration of rotenone, antimycin A, or both in a single step. Validity of using rotenone only for inducing ROX in intact cells is evaluated by stepwise measurement of ROX after titration of rotenone (3Rot) followed by titration of animycin A (4ceAma).
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 titration of animycin 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.
  • 1ce: ROUTINE (R; routine state in cell-culture medium) in intact cells (ce);
  • 2ceOmy: LEAK (L; inhibition of ATPsynthase by 1 μg/ml oligomycin);
  • 3ceU: ET-pathway (E; maximal stimulation by uncoupling of oxidative phosphorylation in four subsequent titrations of the uncoupler, U, FCCP (2.5–4 μM final concentration);
  • 4ceRot: ROX (rotenone, Rot; residual oxygen consumption).
  • 5ceAma: ROX (antimycin A, Ama; residual oxygen consumption). Modified after Huetter_2004_Biochem J.
  • 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. Biochem J 380:919-28. - »Bioblast link«


MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry