SUIT-006 O2 mt D022: Difference between revisions
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|info='''A''': Protocol for coupling control assessment in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) '''[[SUIT-006]]''' | |info='''A''': Protocol for coupling control assessment in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) '''[[SUIT-006]]''' | ||
|application=O2 | |application=O2 | ||
|SUIT number=D022_1SRot;2D;2c; | |SUIT number=D022_1SRot;2D;2c;(3Omy);4U;5Ama | ||
}} | }} | ||
::: '''[[MitoPedia: SUIT]]'''ย ย | ::: '''[[MitoPedia: SUIT]]'''ย ย | ||
::: '''[[Categories of SUIT protocols |SUIT-category]]:''' S | ::: '''[[Categories of SUIT protocols |SUIT-category]]:''' S | ||
::: '''[[SUIT protocol pattern]]:''' linear [[coupling control protocol]] 1SRot;2D;2c;4U;5Ama | ::: '''[[SUIT protocol pattern]]:''' linear [[coupling control protocol]] 1SRot;2D;2c;(3Omy);4U;5Ama | ||
SUIT-006 O2 mt D022 is a [[coupling control protocol]] for [[mitochondrial preparations]] (tissue homogenate, isolated mitochondria and permeabilized cells). Different coupling control states are assessed ([[LEAK-respiration|''L''(n)]] - [[Oxidative phosphorylation| ''P'']] - [[ET-capacity| ''E'']]) at [[Succinate pathway control state|succinate pathway control state]]. The use of [[rotenone]], that inhibits complex I, provides a specific coupling control protocol for S-pathway, avoiding [[Reverse electron flow from CII to CI|reverse electron transfer (RET)]]. ย | SUIT-006 O2 mt D022 is a [[coupling control protocol]] for [[mitochondrial preparations]] (tissue homogenate, isolated mitochondria and permeabilized cells). Different coupling control states are assessed ([[LEAK-respiration|''L''(n)]] - [[Oxidative phosphorylation| ''P'']] - [[ET-capacity| ''E'']]) at [[Succinate pathway control state|succinate pathway control state]]. The use of [[rotenone]], that inhibits complex I, provides a specific coupling control protocol for S-pathway, avoiding [[Reverse electron flow from CII to CI|reverse electron transfer (RET)]]. ย | ||
In the DatLab software, the DLP file for SUIT-006 O2 mt D022 is provided without addition of [[oligomycin]]. For using this protocol with oligomycin addition, a [[Export DL-Protocol User (*.DLPU)|DLPU]] can be created, with the Omy event and mark. Using oligomycin in the | In the DatLab software, the DLP file for SUIT-006 O2 mt D022 is provided without the addition of [[oligomycin]]. For using this protocol with oligomycin addition, a [[Export DL-Protocol User (*.DLPU)|DLPU]] can be created, with the Omy event and mark. Using oligomycin in the preliminary assays with a not yet studied model is useful to determine whether in this kind of sample LEAK respiration in the absence of ADP (1SRot) may be overestimated due to the presence of residual and endogenous adenylates. | ||
Revision as of 11:38, 5 April 2019
Description
Abbreviation: S
Reference: A: Protocol for coupling control assessment in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) SUIT-006
SUIT number: D022_1SRot;2D;2c;(3Omy);4U;5Ama
O2k-Application: O2
- MitoPedia: SUIT
- SUIT-category: S
- SUIT protocol pattern: linear coupling control protocol 1SRot;2D;2c;(3Omy);4U;5Ama
SUIT-006 O2 mt D022 is a coupling control protocol for mitochondrial preparations (tissue homogenate, isolated mitochondria and permeabilized cells). Different coupling control states are assessed (L(n) - P - E) at succinate pathway control state. The use of rotenone, that inhibits complex I, provides a specific coupling control protocol for S-pathway, avoiding reverse electron transfer (RET).
In the DatLab software, the DLP file for SUIT-006 O2 mt D022 is provided without the addition of oligomycin. For using this protocol with oligomycin addition, a DLPU can be created, with the Omy event and mark. Using oligomycin in the preliminary assays with a not yet studied model is useful to determine whether in this kind of sample LEAK respiration in the absence of ADP (1SRot) may be overestimated due to the presence of residual and endogenous adenylates.
Communicated by Cardoso LH, Komlodi T, Huete-Ortega M, Iglesias-Gonzalez J, Doerrier C and Gnaiger E (last update 2019-04-02)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1SRot | SL | S | CII | 1SRot
|
2D | SP | S | CII | 1SRot;2D
|
2c | ScP | S | CII | 1SRot;2D;2c
|
(3Omy) | ScL(Omy) | 1SRot;2D;2c;(3Omy)
| ||
4U | SE | S | CII | 1SRot;2D;2c;4U
|
5Ama | ROX | 1SRot;2D;2c;4U;5Ama
|
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- 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 is recommended when the aim is to analyse coupling control in mtprep (see MitoPedia: Sample preparations) at Succinate pathway control state.
- + The combination of succinate with rotenone, that inhibits complex I, provides the best coupling control protocol specific for S-pathway, avoiding reverse electron transfer (RET).
- + Reasonable duration of the experiment.
- + This protocol can be extended with the Complex IV module.
- - This protocol does not include internal ET-pathway control steps.
- - Careful washing is required after the experiment to avoid carry-over of uncoupler and inhibitors.
Compare SUIT protocols
References
Year | Reference | Organism | Tissue;cell | |
---|---|---|---|---|
Krumschnabel 2014 Methods Enzymol | 2014 | Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81. https://doi.org/10.1016/B978-0-12-416618-9.00009-1 | Mouse | Nervous system |
MitoPedia concepts:
SUIT protocol,
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MitoPedia methods:
Respirometry