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: D059_ce1;ce2SOD;ce3HRP;ce4DMSO;ce5Omy;ce6U;ce7Rot;ce8Ama
O2k-Application: AmR
- MitoPedia: SUIT
- SUIT-category: ce
- SUIT protocol pattern: linear cell (ce) coupling control protocol ce1;ce2Omy;ce3U
SUIT-003 AmR ce D059 is a control protocol of SUIT-003 AmR ce D058 to study the effect of the carrier titrations on the respiration in a parallel experiment.
SUIT-003 AmR ce D059 and SUIT-003 AmR ce D058 were designed to study the inhibitory effect of the components of 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 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 respiration and the ET-capacity. The inhibitory effect of Amplex UltraRed should be tested with each sample type. SUIT-003 AmR ce D058 shows the titration of the AmR assay, while SUIT-003 AmR ce D059 serves as a carrier control titration; these protocols should be run simultaneously.
Communicated by Garcia-Souza LF (last update 2019-05-20)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
ce1 | ROUTINE | ce1
| ||
ce1H2O | ROUTINE | ce1;ce1H2O
| ||
ce1MiR05 | ROUTINE | ce1;ce1H2O;ce1MiR05
| ||
ce1DMSO | ROUTINE | ce1;ce1H2O;ce1MiR05;ce1DMSO
| ||
ce2Omy | LOmy | ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy
| ||
ce3U | E | ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U
| ||
ce4Rot | ROX | ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U;ce4Rot
| ||
ce5Ama | ROX | ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U;ce4Rot;ce5Ama
|
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- Coupling control
- Pathway control
- Β» Electron transfer pathway
- Β» Fatty acid oxidation pathway control state, F
- Β» NADH electron transfer-pathway state, N
- Β» Succinate pathway control state, S
- Β» NS-pathway control state, NS
- Β» Glycerophosphate pathway control state, Gp
- Β» Complex IV single step, CIV
- Β» Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- Β» Glutamate, G
- Β» Glycerophosphate, Gp
- Β» Malate, M
- Β» Octanoylcarnitine, Oct
- Β» Pyruvate, P
- Β» 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 different 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 AmR ce D058: is a protocol to study the inhibitroy effect of the AmR assay towards the respiration
- 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
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 | KomlΓ³di 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