SUIT-003 O2 ce D050: Difference between revisions
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{{MitoPedia | {{MitoPedia | ||
|abbr= CCP-ce Snv | |abbr=CCP-ce Snv | ||
|description=[[File:ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama.png|400px]] | |description=[[File:ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama.png|400px]] | ||
|info='''A:''' - [[Coupling control protocol]] for [[living cells]] with plasma membrane-permeable succinate ([[MitoKit-CII/Succinate-nv]]) - '''[[SUIT-003]]''' | |info='''A:''' - [[Coupling-control protocol]] for [[living cells]] with plasma membrane-permeable succinate ([[MitoKit-CII/Succinate-nv]]) - '''[[SUIT-003]]''' - Plasma membrane-permeable succinate (NV118) in ROUTINE respiration. [[Coupling-control protocol]] (CCP) in living cells (ce) | ||
|application=O2 | |application=O2 | ||
|SUIT number=D050_ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama | |SUIT number=D050_ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama | ||
}} | }} | ||
: | {{Template:SUIT text D050}} | ||
__TOC__ | |||
Communicated by [[Doerrier C]] (last update 2019-09-24) | |||
== Representative traces == | |||
[[File:D050_O2_traces.png|600px]] | |||
{{Template:SUIT-003 O2 ce D050}} | {{Template:SUIT-003 O2 ce D050}} | ||
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== Strengths and limitations == | == Strengths and limitations == | ||
:::* A [[Carrier control titrations|carrier control]] with DMSO is recommended, [[SUIT-003 O2 ce D062]] is provided with this purpose. | :::* A [[Carrier control titrations|carrier control]] with DMSO is recommended, [[SUIT-003 O2 ce D062]] is provided with this purpose. | ||
:::+ | :::+ [[MitoKit-CII/Succinate-nv]] enables the study of succinate-linked respiration, or can overcome a CI-deficiency in [[living cells]]. | ||
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:::+ Reasonable duration of the experiment. | :::+ Reasonable duration of the experiment. | ||
:::+ This is a cell [[coupling control protocol]] that provides information on [[ROUTINE]] and [[ | :::+ This is a cell [[coupling-control protocol]] that provides information on [[ROUTINE]] and [[Electron transfer pathway]] in the presence of ([[MitoKit-CII/Succinate-nv]]. | ||
:::- This protocol is provided without [[oligomycin]] addition. To also include [[LEAK]] in the protocol, an event for the addition of oligomycin and a related mark can be added by editing the DLP file, creating a [[Export DL-Protocol User (*.DLPU)|DLPU]]. However, the addition of [[oligomycin]] has to be carefully tested to avoid inhibitory effects in the evaluation of the [[ET | :::- This protocol is provided without [[oligomycin]] addition. To also include [[LEAK respiration]] in the protocol, an event for the addition of oligomycin and a related mark can be added by editing the DLP file, creating a [[Export DL-Protocol User (*.DLPU)|DLPU]]. However, 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. | :::- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler. | ||
== Compare SUIT protocols == | == Compare SUIT protocols == | ||
::::* [[SUIT-003 O2 ce D062]] [[Carrier control titrations|carrier control]] SUIT protocol for SUIT-003 O2 ce D050 in living cells. The carrier of the plasma membrane-permeable succinate (DMSO) is added in ROUTINE. ย | ::::* [[SUIT-003 O2 ce D062]] [[Carrier control titrations|carrier control]] SUIT protocol for SUIT-003 O2 ce D050 in living cells. The carrier of the plasma membrane-permeable succinate (DMSO) is added in ROUTINE. ย | ||
::::* [[SUIT-003 O2 ce D060]] CCP with cell permeable compounds (MitoKit-CII/Succinate-nv and MitoKit-CII/Malonate-nv). | ::::* [[SUIT-003 O2 ce D060]] CCP with cell-permeable compounds (MitoKit-CII/Succinate-nv and MitoKit-CII/Malonate-nv). | ||
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== Chemicals and syringes == | |||
{{Template:Chemicals SUIT-003 D050}} | |||
::: Suggested stock concentrations are shown in the specific DL-Protocol. | |||
== References == | == References == | ||
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|order=descending | |order=descending | ||
}} | }} | ||
{{MitoPedia concepts | {{MitoPedia concepts | ||
|mitopedia concept=SUIT protocol, SUIT A, Find | |mitopedia concept=SUIT protocol, SUIT A, Find | ||
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|mitopedia method=Respirometry | |mitopedia method=Respirometry | ||
}} | }} | ||
{{MitoPedia O2k and high-resolution respirometry}} | |||
{{MitoPedia topics}} |
Latest revision as of 11:04, 26 April 2021
Description
Abbreviation: CCP-ce Snv
Reference: A: - Coupling-control protocol for living cells with plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) - SUIT-003 - Plasma membrane-permeable succinate (NV118) in ROUTINE respiration. Coupling-control protocol (CCP) in living cells (ce)
SUIT number: D050_ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama
O2k-Application: O2
SUIT-003 O2 ce D050 protocol is designed to study CI-deficiency in living cells. Plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv, Snv), which is added in ROUTINE stimulates mitochondrial respiration in CI-deficient human blood cells, fibroblasts and heart fibres, acting on Complex II of the electron transfer system. Moreover, the SUIT-003 O2 ce D050 protocol enables evaluation of the coupling control of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE and Electron transfer pathway. Optionally, 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 (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
| ||
ce1Snv | ROUTINE | ce1
| ||
(ce2Omy) | L(Omy) | ce1;ce1Snv;(ce2Omy)
| ||
ce3U | E | ce1;ce1Snv;(ce2Omy);ce3U
| ||
ce4Rot | ROX | ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot
| ||
ce5Ama | ROX | ce1;ce1Snv;(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
- A carrier control with DMSO is recommended, SUIT-003 O2 ce D062 is provided with this purpose.
- + MitoKit-CII/Succinate-nv enables the study of succinate-linked respiration, or can overcome a CI-deficiency in living cells.
- + Reasonable duration of the experiment.
- + This is a cell coupling-control protocol that provides information on ROUTINE and Electron transfer pathway in the presence of (MitoKit-CII/Succinate-nv.
- - This protocol is provided without oligomycin addition. To also include LEAK respiration in the protocol, an event for the addition of oligomycin and a related mark can be added by editing the DLP file, creating a DLPU. However, 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 D062 carrier control SUIT protocol for SUIT-003 O2 ce D050 in living cells. The carrier of the plasma membrane-permeable succinate (DMSO) is added in ROUTINE.
- SUIT-003 O2 ce D060 CCP with cell-permeable compounds (MitoKit-CII/Succinate-nv and MitoKit-CII/Malonate-nv).
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
ce1Snv | Succinate-nv (Snv) | |
(ce2Omy) | Oligomycin (Omy) | This step can be skipped |
ce3U | Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) | Can be substituted for other uncoupler |
ce4Rot | Rotenone (Rot) | |
ce5Ama | Antimycin A (Ama) |
- Suggested stock concentrations are shown in the specific DL-Protocol.
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Respirometry