SUIT-003 AmR ce D058: Difference between revisions
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::: '''[[SUIT protocol pattern]]:'''ce1;ce2SOD;ce3HRP;ce4AmR;ce5Omy;ce6U;ce7Rot;ce8Ama | ::: '''[[SUIT protocol pattern]]:'''ce1;ce2SOD;ce3HRP;ce4AmR;ce5Omy;ce6U;ce7Rot;ce8Ama | ||
SUIT-003 O2 ce D058 has been designed to analyse the inhibitory effect the Amplex UltraRed assay on [[coupling control state]] of living 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 further 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 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. [[SUIT-003 AmR ce D059]] should be performed simultaneously as a [[carrier control titration]]. | SUIT-003 O2 ce D058 has been designed to analyse the inhibitory effect the Amplex UltraRed assay on [[coupling control state]] of living 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 further 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 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. [[SUIT-003 AmR ce D059]] should be performed simultaneously as a [[carrier control titrations| carrier control titration]]. | ||
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Revision as of 17:00, 24 June 2019
Description
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 inhibitory effect the Amplex UltraRed assay on coupling control state of living 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 further 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 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. 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
|
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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 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 | Komlรณdi 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 |
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 |
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 |
MitoPedia concepts: SUIT protocol, SUIT B, Find
MitoPedia methods:
Respirometry