Difference between revisions of "SUIT-009 AmR mt D021"
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== Strengths and limitations == | == Strengths and limitations == | ||
::::* It a protocol to investigate: | ::::* It is a protocol to investigate: | ||
::::::::* the S- and NS-pathway linked respiration and ROS production. | ::::::::* the S- and NS-pathway linked respiration and ROS production. | ||
::::::::* any Complex I defect, which would lead to decreased reverse electron transfer supported ROS production. | ::::::::* any Complex I defect, which would lead to decreased reverse electron transfer supported ROS production. | ||
::::::::* any Complex II defect, which would result is decreased S-linked respiration and ROS production. | ::::::::* any Complex II defect, which would result is decreased S-linked respiration and ROS production. | ||
::: + You can investigate in the absence of ADP the reverse electron transfer supported H<sub>2</sub>O<sub>2</sub> production in S respiring mitochondria,which will be inhibited by ADP owing to the depolarisation of the mt-membrane. | |||
::: + Reasonable duration of the experiment. | |||
:::*[[SUIT-009 O2 mt D015]] determination of O<sub>2</sub> flux on isolated mitochondria and tissue homogenate as a control for SUIT-009 AmR mt D021 to determine the inhibitory effect of the fluorescence dye on the mt-respiration. | :::*[[SUIT-009 O2 mt D015]] determination of O<sub>2</sub> flux on isolated mitochondria and tissue homogenate as a control for SUIT-009 AmR mt D021 to determine the inhibitory effect of the fluorescence dye on the mt-respiration. | ||
:::+ You can investigate in the absence of ADP the reverse electron transfer supported H2O2 production in S respiring mitochondria,which will be inhibited by ADP owing to the depolarisation of the mt-membrane. | :::+ You can investigate in the absence of ADP the reverse electron transfer supported H2O2 production in S respiring mitochondria,which will be inhibited by ADP owing to the depolarisation of the mt-membrane. |
Revision as of 11:08, 12 February 2019
Description
Abbreviation: NS(P)
Reference: A: SUIT-009 short protocol for H2O2 (AmR) in mitochondrial preparations (isolated mitochondria and tissue homogenate).
SUIT number: D021_1S;2D;3P;4Rot;5Ama
O2k-Application: AmR
- SUIT-category: NS(P)
- SUIT protocol pattern: orthogonal 1S;2D;3P;4Rot;5Ama
SUIT 9 is a short protocol for simultaneous determination of O2 flux and the rate of H2O2 production (H2O2 flux) on isolated mitochondria,tissue homogenate (except of liver) and permeabilized cells. Succinate (S) supports the reverse electron transfer (RET) initiated H2O2 production in the LEAK state. Addition of ADP decreases H2O2 flux due to depolarization of the mitochondrial membrane. Pyruvate (P) supports NADH-pathway (N), and usually in the presence of ADP it does not increase further the H2O2 flux. Antimycin A (Ama) inhibits Complex III (CIII) and results in the elevation of the H2O2 flux. The sensitivity of the Amplex UltraRed® assay (for determining H2O2 production) is changing over the experimental time and upon addition of chemicals. To correct H2O2 flux for the sensitivity changes several H2O2 calibration steps are done during the experiment. The measurement is carried out in MiR05-Kit at 37 °C. For O2-Application, choose SUIT-009 O2 mt D015.
Communicated by Iglesias-Gonzalez J, Komlodi T and Gnaiger E (last update 2019-02-12)
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 |
Year | Reference | Organism | Tissue;cell | |
---|---|---|---|---|
Chang 2018 Life Sciences Meeting 2018 Innsbruck AT | 2018 | pH dependence of mitochondrial respiration and H2O2 production in oral cancer cells – a pilot study. | Human | HEK Kidney |
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) |
---|---|---|---|---|
1S | SL(n) | S | CII | 1S
|
2D | SP | S | CII | 1S;2D
|
3P | NSP | NS | CI&II | 1S;2D;3P
|
4Rot | SP | S | CII | 1S;2D;3P;4Rot
|
5Ama | ROX | 1S;2D;3P;4Rot;5Ama
|
- 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
- It is a protocol to investigate:
- the S- and NS-pathway linked respiration and ROS production.
- any Complex I defect, which would lead to decreased reverse electron transfer supported ROS production.
- any Complex II defect, which would result is decreased S-linked respiration and ROS production.
- + You can investigate in the absence of ADP the reverse electron transfer supported H2O2 production in S respiring mitochondria,which will be inhibited by ADP owing to the depolarisation of the mt-membrane.
- + Reasonable duration of the experiment.
- SUIT-009 O2 mt D015 determination of O2 flux on isolated mitochondria and tissue homogenate as a control for SUIT-009 AmR mt D021 to determine the inhibitory effect of the fluorescence dye on the mt-respiration.
- + You can investigate in the absence of ADP the reverse electron transfer supported H2O2 production in S respiring mitochondria,which will be inhibited by ADP owing to the depolarisation of the mt-membrane.
- - CIV activity and Cytochrome c test cannot be performed together with the fluorescence.
Compare SUIT protocols
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Fluorometry