Difference between revisions of "SUIT-003 AmR ce D058"
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|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]]''' | |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]]''' | ||
}} | }} | ||
::: '''[[SUIT protocol pattern]]:'''ce1;ce2SOD;ce3HRP;ce4AmR;ce5Omy;ce6U;ce7Rot;ce8Ama | ::: '''[[SUIT protocol pattern]]:'''ce1;ce2SOD;ce3HRP;ce4AmR;ce5Omy;ce6U;ce7Rot;ce8Ama | ||
Revision as of 11:42, 25 June 2019
Description
Abbreviation: AmR effect on living cells
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 protocol pattern:ce1;ce2SOD;ce3HRP;ce4AmR;ce5Omy;ce6U;ce7Rot;ce8Ama
SUIT-003 O2 ce D058 has been designed to analyse the possible inhibitory effect of the Amplex UltraRed assay on the living cells coupling control state. Usually, the Superoxide dismutase and the Horseradish peroxidase do not show any inhibitory effect over the respiration. However, the Amplex UltraRed seems to inhibit ROUTINE respiration and the ET-capacity at high concentrations. For this reason, the inhibitory effect of Amplex UltraRed should be tested with each type of sample used prior to the study of ROS production. SUIT-003 AmR ce D059 should be performed simultaneously as a carrier control titration.
Communicated by Komlodi T, Garcia-Souza LF and Gnaiger E (last update 2019-06-24)
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
- Main fuel substrates
- » Glutamate, G
- » Glycerophosphate, Gp
- » Malate, M
- » Octanoylcarnitine, Oct
- » Pyruvate, P
- » Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
- This protocol has been designed to study the inhibitory effect of the Amplex UltraRed assay over living cells.
- It is recommended to run this test with every different sample type used.
- + 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 AmR ce D059 is a control protocol of to study the effect of the carrier titrations on the respiration in a parallel experiment.
- SUIT-003 O2 ce D009 for non-permeabilized cells with oligomycin without the AmR assay
References
Year | Reference | Organism | Tissue;cell | |
---|---|---|---|---|
MiPNet24.10 H2O2 flux analysis | 2021-10-22 | Hydrogen peroxide flux analysis using Amplex UltraRed assay in MiR05-Kit with DatLab 7.4 | ||
Komlodi 2021 BEC AmR-O2 | 2021 | Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. Bioenerg Commun 2021.4. https://doi.org/10.26124/bec:2021-0004 | Saccharomyces cerevisiae | Other cell lines |
MiPNet20.14 AmplexRed H2O2-production | 2019-06-24 | O2k-FluoRespirometry: HRR and simultaneous determination of H2O2 production with Amplex UltraRed. | Mouse | Heart |
Komlodi 2018 Methods Mol Biol | 2018 | Komlodi T, Sobotka O, Krumschnabel G, Bezuidenhout N, Hiller E, Doerrier C, Gnaiger E (2018) Comparison of mitochondrial incubation media for measurement of respiration and hydrogen peroxide production. Methods Mol Biol 1782:137-55. | Human Mouse | Skeletal muscle HEK |
Krumschnabel 2015 Methods Mol Biol | 2015 | Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61. | Mouse | Nervous system |
Makrecka-Kuka 2015 Biomolecules | 2015 | Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. https://doi.org/10.3390/biom5031319 | Human Mouse | Heart Nervous system HEK |
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Respirometry