SUIT-003 O2 ce D009: Difference between revisions
No edit summary |
No edit summary ย |
||
(47 intermediate revisions by 9 users not shown) | |||
Line 1: | Line 1: | ||
{{MitoPedia | {{MitoPedia | ||
|abbr=ce | |abbr= CCP-ce short | ||
|description=[[File:1ce;2ceOmy;3ceU-.jpg| | |description=[[File:1ce;2ceOmy;3ceU-.jpg|450px]] | ||
|info='''A''' [[ | |info='''A:''' - '''[[SUIT-003]]''' - [[Coupling-control protocol]] (CCP) in living cells (ce) with oligomycin (LEAK state) | ||
|application=O2 | |application=O2 | ||
|SUIT number=D009_ce1;ce2Omy;ce3U; | |SUIT number=D009_ce1;ce2Omy;ce3U;ce4Ama | ||
}} | }} | ||
: | ย | ||
{{Template:SUIT text D009}} | |||
ย | |||
__TOC__ | __TOC__ | ||
{{Template:SUIT-003 O2 O2 ce D009}} | Communicated by [[Doerrier C]], [[Cardoso LHD]], [[Gnaiger E]] (last update 2019-09-24) | ||
ย | |||
== Representative traces == | |||
ย | |||
[[File:D009 Traces.png|600px]] | |||
ย | |||
{{Template:SUIT-003 O2 ce D009}} | |||
ย | |||
== 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 (''N''<sub>ce</sub>) is the sum of viable cells (''N''<sub>vce</sub>) and dead cells (''N''<sub>dce</sub>). | |||
:::* 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. | |||
ย | |||
== 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;ce1P;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). | |||
ย | |||
== Chemicals and syringes == | |||
{{Template:Chemicals SUIT-003}} | |||
::: Suggested stock concentrations are shown in the specific DL-Protocol. | |||
== References == | == References == | ||
Line 23: | Line 54: | ||
|order=descending | |order=descending | ||
}} | }} | ||
[[File:1ce;2ceOmy;3ceU;4ceRot;5ceAma.jpg|right|200px]] | [[File:1ce;2ceOmy;3ceU;4ceRot;5ceAma.jpg|right|200px]] | ||
Line 36: | Line 60: | ||
:::* 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 | :::: 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). | ||
[[File:Huetter 2004 Biochem J.jpg|left|300px|ceCCP (fibroblasts)]] | [[File:Huetter 2004 Biochem J.jpg|left|300px|ceCCP (fibroblasts)]] | ||
<br /> | <br /> | ||
ย | :::: Respiration in senescent human primary fibroblasts (0.2ร10<sup>6</sup> cells/ml). Arrows show steps in the titration regime of the [[coupling-control protocol]], inducing the following respiratory states. ย | ||
:::: Respiration in senescent human primary fibroblasts (0.2ร10<sup>6</sup> cells/ml). Arrows show steps in the titration regime of the [[coupling control protocol]], inducing the following respiratory states. ย | ::::* ce1: ROUTINE (''R''; routine state in cell-culture medium) in living cells (ce); ย | ||
::::* | ::::* ce2Omy: LEAK (''L''; inhibition of ATP-synthase by 1 ฮผg/mL oligomycin); ย | ||
::::* | ::::* ce3U: ET-state (''E''; maximal stimulation by uncoupling of oxidative phosphorylation in four subsequent titrations of the uncoupler, U, FCCP (2.5โ4 ฮผM final concentration); ย | ||
::::* | ::::* ce4Rot: ROX (rotenone, Rot; residual oxygen consumption); | ||
::::* | ::::* ce5Ama: ROX (antimycin A, Ama; residual oxygen consumption). Modified after [[Huetter_2004_Biochem J]]. | ||
::::* | |||
ย | |||
{{MitoPedia concepts | {{MitoPedia concepts |
Latest revision as of 23:48, 12 April 2021
Description
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
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
- ยป Electron transfer pathway
- ยป Fatty acid oxidation pathway control state, F
- ยป NADH electron transfer-pathway state, N
- ยป Succinate pathway control state, S
- ยป NS-pathway control state, NS
- ยป Glycerophosphate pathway control state, Gp
- ยป Complex IV single step, CIV
- ยป Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- ยป Glutamate, G
- ยป Glycerophosphate, Gp
- ยป Malate, M
- ยป Octanoylcarnitine, Oct
- ยป Pyruvate, P
- ยป Succinate, S
- Main fuel substrates
- Glossary
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
- 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;ce1P;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).
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.
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 | Hรผtter E, Renner K, Pfister G, Stรถckl P, Jansen-Dรผrr 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 living 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 (0.2ร106 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 ฮผg/mL oligomycin);
- ce3U: ET-state (E; maximal stimulation by uncoupling of oxidative phosphorylation in four subsequent titrations of the uncoupler, U, FCCP (2.5โ4 ฮผM 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 (0.2ร106 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,
Find
MitoPedia methods:
Respirometry