Description
Abbreviation: CCP(P) and cell membrane permeability test
Reference: B - SUIT-003
SUIT number: D017_ce1;ce2P;ce3Omy;ce4U;ce5Rot;ce6S;ce7Ama
O2k-Application: O2
- MitoPedia: SUIT - coupling control protocol (CCP) with pyruvate and cell membrane permeability test
- SUIT protocol pattern: ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama
This protocol has been designed to study the H2O2 production in various coupling control states and test the cell membrane permeability with the same protocol using living cells. Pyruvate is added to support the ROUTINE respiration of living cells when they are in a respiratory for mitochondrial preparations (i.e. MiR05). To initiate LEAK-respiration oligomycin is added, inhibiting the ATP-synthase and leading to an increased in the H2O2 production. Whereas titrations of uncoupler results in a decreased H2O2 generation. However, one of the main problems with living cells is that the H2O2 flux is very low or hardly detectable due to the scavenger activity of the antioxidant enzymes in the cytosol.
Communicated by Komlodi T, Doerrier C, Iglesias-Gonzalez J and Cardoso LH (last update 2019-06-24)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
0DTPA |
| |||
0SOD |
| |||
0HRP |
| |||
0AmR |
|
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
ce1 | ROUTINE | ce1
| ||
ce1P | ROUTINE | ce1;ce1P; ROUTINE, R
| ||
ce2Omy | L(Omy) | ce1;ce1P;ce2Omy
| ||
ce3U | E | ce1;ce1P;ce2Omy;ce3U
| ||
ce4Rot | ROX | ce1;ce1P;ce2Omy;ce3U;ce4Rot
| ||
ce5S | ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S
| |||
ce6Ama | ROX | ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama
|
Strengths and limitations
- When MiR05 is used, the addition of pyruvate is recommended to provide an external fuel substrate.
- When culture medium (e.g. DMEM) is used, application of phenol red is not advisable, since it can interfere with the fluorescence signal.
- + Reasonable duration of the experiments.
- - Minor changes in the H2O2 flux can be seen with living cells because of the action of the antioxidant enzymes in the cytosol, which scavenge the H2O2 produced. (AmR is not permeable to the cell membrane, therefore, only the H2O2 that crosses the plasma membrane can be measured)
Compare SUIT protocols
- SUIT-013 AmR ce D023: SUIT-013 has been designed to study the oxygen dependence of H2O2 production in intact cells.
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 |
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 B,
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MitoPedia methods:
Fluorometry