SUIT-003 AmR ce D058: Difference between revisions
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{{MitoPedia | {{MitoPedia | ||
|abbr=ce | |abbr=ce | ||
|description= | |description=[[File:1ce;2ceOmy;3ceU-.jpg|150px]] | ||
|info='''B:''' Coupling control in intact cells with oligomycin; ce: non-permeabilized cells -'''[[SUIT-003]]''' | |info='''B:''' Coupling control in intact cells with oligomycin to study the inhibitory effect of the AmR assay components on the respiration ; ce: non-permeabilized cells -'''[[SUIT-003]]''' ย | ||
|application= | |application=AmR | ||
|SUIT number= | |SUIT number=D058_ce1;ce2Omy;ce3U;ce4Rot;ce5Ama | ||
}} | }} | ||
::: '''[[MitoPedia: SUIT]]''' | ::: '''[[MitoPedia: SUIT]]''' | ||
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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 | SUIT-003 O2 ce D058 has been designed to study the inhibitory effect of the components of the Amplex UltraRed assay on [[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-pathway|electron transfer system]], has to be studied to avoid an underestimation of the [[ET-capacity]]. [[Superoxide dismutase]] and [[Horseradish peroxidase]] do not inhibit respiration, however, [[Amplex UltraRed]] seems to block at higher concentration [[ROUTINE]] respiratoin and the [[ET-capacity]]. The inhibitory effect of [[Amplex UltraRed]] should be tested with each sample type. ย | ||
__TOC__ | __TOC__ | ||
ย Communicated byย [[Komlodi T]] and [[Gnaiger E]] (last update 2019-05- | ย Communicated byย [[Komlodi T]] and [[Gnaiger E]] (last update 2019-05-15) | ||
{{Template:SUIT-003 AmR ce D058}} | {{Template:SUIT-003 AmR ce D058}} | ||
== Strengths and limitations == | == Strengths and limitations == | ||
:::* | :::* This protocol has been designed to study the inhibitory effect of the Amplex UltraRed assay. ย | ||
:::* | :::* It is recommended to run this test with every differetn sample type. | ||
:::+ Reasonable duration of the experiment. | :::+ 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]]. | :::- The concentration of [[oligomycin]] has to be carefully tested to avoid inhibitory effects in the evaluation of the [[ET-capacity]]. | ||
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== Compare SUIT protocols == | == Compare SUIT protocols == | ||
::::* [[SUIT-003 O2 ce | ::::* [[SUIT-003 O2 ce D009]] for non-permeabilized cells with oligomycin without the AmR assay | ||
::::* | ::::* Protocols using AmR assay: | ||
::::* [[SUIT- | :::::* [[SUIT-006 AmR mt D048]] to study simultaneously the O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> flux on isolated mitochondria, tissue homogenate and permeabilized cells, in particular to study the influence of mt-membrane potential on NADH-linked pathway initiated mt-H2O2 production. | ||
::::* [[SUIT- | :::::*[[SUIT-009 AmR ce-pce D019]] simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux for non-permeabilized (ce) and permeabilized cells (pce; permeabilization of the plasma membrane occurs in the chambers through addition of digitonin ) | ||
:::::* [[SUIT-009 AmR mt D021]] simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux on isolated mitochondria and tissue homogenate | |||
::::* [[SUIT- | :::::* [[SUIT-013 AmR ce D023]] for intact cells | ||
::::* [[SUIT- | :::::*[[SUIT-018 AmR mt D031]] simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux on isolated mitochondria, tissue homogenate and permeabilized cells at low oxygen concentration (tissue normoxia). | ||
::::* [[SUIT- | :::::* [[SUIT-018 AmR mt D040]] simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux on isolated mitochondria, tissue homogenate and permeabilized cells at high oxygen concentration (tissue hyperoxia). | ||
::::* [[SUIT- | :::::* [[SUIT-018 AmR mt D041]] simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux on isolated mitochondria, tissue homogenate and permeabilized cells changing the oxygen concentration in the same protocol. ย | ||
::::* [[SUIT- | |||
== References == | == References == | ||
{{#ask:[[Category:Publications]] [[Additional label::SUIT-003 O2 ce D009]] | {{#ask:[[Category:Publications]] [[Additional label::SUIT-003 O2 ce D009]] [[Additional label::SUIT-003 AmR ce D058]] | ||
|?Was published in year=Year | |?Was published in year=Year | ||
|?Has title=Reference | |?Has title=Reference | ||
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[[File:1ce;2ceOmy;3ceU;4ceRot;5ceAma.jpg|right|200px]] | [[File:1ce;2ceOmy;3ceU;4ceRot;5ceAma.jpg|right|200px]] | ||
{{MitoPedia concepts | {{MitoPedia concepts |
Revision as of 10:25, 15 May 2019
Description
Abbreviation: ce
Reference: B: Coupling control in intact cells with oligomycin to study the inhibitory effect of the AmR assay components on the respiration ; ce: non-permeabilized cells -SUIT-003
SUIT number: D058_ce1;ce2Omy;ce3U;ce4Rot;ce5Ama
O2k-Application: AmR
- MitoPedia: SUIT
- SUIT-category: ce
- SUIT protocol pattern: linear cell (ce) coupling control protocol ce1;ce2Omy;ce3U
SUIT-003 O2 ce D058 has been designed to study the inhibitory effect of the components of the Amplex UltraRed assay on 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. Superoxide dismutase and Horseradish peroxidase do not inhibit respiration, however, Amplex UltraRed seems to block at higher concentration ROUTINE respiratoin and the ET-capacity. The inhibitory effect of Amplex UltraRed should be tested with each sample type.
Communicated by Komlodi T and Gnaiger E (last update 2019-05-15)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
ce1 | ROUTINE | ce1
| ||
ce1SOD | ROUTINE | ce1;ce1SOD
| ||
ce1HRP | ROUTINE | ce1;ce1SOD;ce1HRP
| ||
ce1AmR | ROUTINE | ce1;ce1SOD;ce1HRP;ce1AmR
| ||
ce2Omy | LOmy | ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy
| ||
ce3U | E | ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U
| ||
ce4Rot | ROX | ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U;ce4Rot
| ||
ce5Ama | ROX | ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U;ce4Rot;ce5Ama
|
- 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
- This protocol has been designed to study the inhibitory effect of the Amplex UltraRed assay.
- It is recommended to run this test with every differetn sample type.
- + 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
- SUIT-003 O2 ce D009 for non-permeabilized cells with oligomycin without the AmR assay
- Protocols using AmR assay:
- SUIT-006 AmR mt D048 to study simultaneously the O2 flux and H2O2 flux on isolated mitochondria, tissue homogenate and permeabilized cells, in particular to study the influence of mt-membrane potential on NADH-linked pathway initiated mt-H2O2 production.
- SUIT-009 AmR ce-pce D019 simultaneous determination of O2 and H2O2 flux for non-permeabilized (ce) and permeabilized cells (pce; permeabilization of the plasma membrane occurs in the chambers through addition of digitonin )
- SUIT-009 AmR mt D021 simultaneous determination of O2 and H2O2 flux on isolated mitochondria and tissue homogenate
- SUIT-013 AmR ce D023 for intact cells
- SUIT-018 AmR mt D031 simultaneous determination of O2 and H2O2 flux on isolated mitochondria, tissue homogenate and permeabilized cells at low oxygen concentration (tissue normoxia).
- SUIT-018 AmR mt D040 simultaneous determination of O2 and H2O2 flux on isolated mitochondria, tissue homogenate and permeabilized cells at high oxygen concentration (tissue hyperoxia).
- SUIT-018 AmR mt D041 simultaneous determination of O2 and H2O2 flux on isolated mitochondria, tissue homogenate and permeabilized cells changing the oxygen concentration in the same protocol.
References
MitoPedia concepts: SUIT protocol, SUIT B, Find
MitoPedia methods:
Respirometry