SUIT-003 AmR ce D017: Difference between revisions
No edit summary |
No edit summary |
||
Line 9: | Line 9: | ||
::: '''[[SUIT protocol pattern]]:''' complex cell [[coupling control protocol]] ce1;ce1P;ce2Omy;ce3U;- | ::: '''[[SUIT protocol pattern]]:''' complex cell [[coupling control protocol]] ce1;ce1P;ce2Omy;ce3U;- | ||
This protocol has been designed to study the H<sub>2</sub>O<sub>2</sub> production in various coupling control states and test the cell membrane permeability with the same protocol.Pyruvate is added to support the ROUTINE respiration with intact cells | This protocol has been designed to study the H<sub>2</sub>O<sub>2</sub> production in various coupling control states and test the cell membrane permeability with the same protocol using intact cells. Pyruvate is added to support the ROUTINE respiration with intact cells. To initiate LEAK-respiration oligomycin was added which inhibits the ATP-synthase leading to increased H<sub>2</sub>O<sub>2</sub> production. Whereas titrations of uncoupler results in decreased H<sub>2</sub>O<sub>2</sub> generation. However, the disadvantage of this protocol that the H<sub>2</sub>O<sub>2</sub> flux is very low or hardly detectable owing to the eliminating action of the anitoxidant enzymes in the cytosol. | ||
Line 19: | Line 19: | ||
== Limitations == | == Limitations == | ||
::::* When MiR05 is used, the addition of pyruvate is recommended to provide an external fuel substrate. | ::::* 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. ย | ::::+ Reasonable duration of the experiments. ย | ||
::::- Minor changes in the H<sub>2</sub>O<sub>2</sub> flux can be seen with intact cells because of the action of the antioxidant enzymes in the cytosol, which eliminate the H<sub>2</sub>O<sub>2</sub> produced. (AmR is not permeable to the cell membrane, therefore, the H<sub>2</sub>O<sub>2</sub> must leave the cytosol.) | ::::- Minor changes in the H<sub>2</sub>O<sub>2</sub> flux can be seen with intact cells because of the action of the antioxidant enzymes in the cytosol, which eliminate the H<sub>2</sub>O<sub>2</sub> produced. (AmR is not permeable to the cell membrane, therefore, the H<sub>2</sub>O<sub>2</sub> must leave the cytosol.) |
Revision as of 14:40, 9 May 2019
Description
Abbreviation: ceN(P)
Reference: B - SUIT-003 coupling control protocol (CCP) with pyruvate and cell membrane permeability test
SUIT number: D017_ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama
O2k-Application: AmR
- SUIT-category: ceN(P)
- SUIT protocol pattern: complex cell coupling control protocol ce1;ce1P;ce2Omy;ce3U;-
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 intact cells. Pyruvate is added to support the ROUTINE respiration with intact cells. To initiate LEAK-respiration oligomycin was added which inhibits the ATP-synthase leading to increased H2O2 production. Whereas titrations of uncoupler results in decreased H2O2 generation. However, the disadvantage of this protocol that the H2O2 flux is very low or hardly detectable owing to the eliminating action of the anitoxidant enzymes in the cytosol.
Communicated by Komlodi T, Doerrier C and Cardoso LH (last update 2019-05-09)
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
|
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 intact cells because of the action of the antioxidant enzymes in the cytosol, which eliminate the H2O2 produced. (AmR is not permeable to the cell membrane, therefore, the H2O2 must leave the cytosol.)
- - The AmR method with intact cells using the SUIT-003 protocol has limitations: Only changes in H2O2 flux after adding cells can be quantitatively evaluated.
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 |
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:
Fluorometry