SUIT-003 O2 ce D009: Difference between revisions
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::: '''[[SUIT protocol pattern]]:''' linear cell (ce) [[coupling control protocol]] ce1;ce2Omy;ce3U | ::: '''[[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]] state, 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]]. Β | 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]] state, 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-pathway|electron transfer system]], has to be studied to avoid an underestimation of the [[ET-capacity]]. Β | ||
Revision as of 09:24, 3 April 2019
Description
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 state, 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)
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*
|
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- 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.
- + Reasonable duration of the experiment.
- - 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.
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 | 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 |
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 (0.2Γ106 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 ΞΌg/ml oligomycin);
- ce3U: ET-state (E; maximal stimulation by uncoupling of oxidative phosphorylation in four subsequent titrations of the uncoupler, U, FCCP (2.5β4 ΞΌM final concentration);
- ce4Rot: ROX (rotenone, Rot; residual oxygen consumption).
- ce5Ama: 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.
MitoPedia concepts:
SUIT protocol,
SUIT A,
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MitoPedia methods:
Respirometry