From Bioblast
Description
Abbreviation: ce
Reference: A: SUIT-003
SUIT number: D009_ce1;ce2Omy;ce3U;ce4Rot;ce5Ama
O2k-Application: O2
- SUIT-category: ce
- SUIT protocol pattern: linear cell (ce) coupling control protocol ce1;ce2Omy;ce3U
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 | HΓΌtter E, Renner K, Pfister G, StΓΆckl P, Jansen-DΓΌrr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. https://doi.org/10.1042/BJ20040095 | Human | Fibroblast |
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
ce1 | ROUTINE | ce1
| ||
ce2Omy | LOmy | ce1;ce2Omy
| ||
ce3U | E | ce1;ce2Omy;ce3U
| ||
ce* | ROX | ce1;ce2Omy;ce3U;ce*
|
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- Coupling control
- Pathway control
- Β» Electron transfer pathway
- Β» Fatty acid oxidation pathway control state, F
- Β» NADH electron transfer-pathway state, N
- Β» Succinate pathway control state, S
- Β» NS-pathway control state, NS
- Β» Glycerophosphate pathway control state, Gp
- Β» Complex IV single step, CIV
- Β» Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- Β» Glutamate, G
- Β» Glycerophosphate, Gp
- Β» Malate, M
- Β» Octanoylcarnitine, Oct
- Β» Pyruvate, P
- Β» Succinate, S
- Main fuel substrates
- Glossary
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
- 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
- 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).
- Respiration in senescent human primary fibroblasts (0.2Γ106 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 ΞΌg/ml oligomycin);
- 3ceU: ET-pathway (E; maximal stimulation by uncoupling of oxidative phosphorylation in four subsequent titrations of the uncoupler, U, FCCP (2.5β4 ΞΌM final concentration);
- 4ceRot: ROX (rotenone, Rot; residual oxygen consumption).
- 5ceAma: ROX (antimycin A, Ama; residual oxygen consumption). Modified after Huetter_2004_Biochem J.
- Respiration in senescent human primary fibroblasts (0.2Γ106 cells/ml). Arrows show steps in the titration regime of the coupling control protocol, inducing the following respiratory states.
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MitoPedia concepts:
SUIT protocol,
SUIT A,
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MitoPedia methods:
Respirometry