Difference between revisions of "SUIT-009 AmR ce-pce D019"
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|abbr=NS(P) | |abbr=NS(P) | ||
|description=[[File:Ce1;1Dig;1S;2D;3P;4Rot;5Ama.png|500px|SUIT9]] | |description=[[File:Ce1;1Dig;1S;2D;3P;4Rot;5Ama.png|500px|SUIT9]] | ||
|info='''A''': '''[[SUIT-009]]''' short protocol for H<sub>2</sub>O<sub>2</sub> (AmR) in [[permeabilized cells |pce]]. | |info='''A''': '''[[SUIT-009]]''' short protocol for H<sub>2</sub>O<sub>2</sub> (AmR) in [[permeabilized cells |pce]]. | ||
|SUIT number=D019_ce1;1Dig;1S;2D;3P;4Rot;5Ama | |SUIT number=D019_ce1;1Dig;1S;2D;3P;4Rot;5Ama |
Revision as of 13:32, 5 March 2019
Description
Abbreviation: NS(P)
Reference: A: SUIT-009 short protocol for H2O2 (AmR) in pce.
SUIT number: D019_ce1;1Dig;1S;2D;3P;4Rot;5Ama
- MitoPedia: SUIT - SUIT reference protocol
- SUIT-category: NS(P)
- SUIT protocol pattern: orthogonal 1S;2D;3P;4Rot;5Ama
SUIT-009 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) promoting the H2O2 production in the LEAK state. 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 could increase the H2O2 flux. The sensitivity of the Amplex UltraRed® assay (for determining H2O2 production) changes 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.
Communicated by Iglesias-Gonzalez J, Komlodi T and Gnaiger E (last update 2019-01-23)
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
| ||
1Dig | Dig | ce1;1Dig
|
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 |
|
- 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 assess:
- the S- and NS-pathway linked respiration and ROS production.
- any Complex I defect, which would lead to a decrease in the N-linked respiration and a reduced reverse electron transfer supported ROS production.
- any Complex II defect, which would result in decreased S-linked respiration and ROS production.
- SUIT-009 O2 pce D016 determination of O2 flux on permeabilized cells as a control for SUIT-009 AmR pce D019 to determine the inhibitory effect of the fluorescent 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 due to the depolarisation of the mt-membrane.
- + Reasonable duration of the experiment.
- - CIV activity and Cytochrome c test cannot be performed together with the fluorescence assay.
- - The H2O2 flux will be much lower compared to that observed with isolated mitochondria, which might be explained by the high antioxidant capacity of the cytosol.
- - In some cell lines e.g. HEK293T, P does not have any effect because of the downregulation of the N-linked pathway.
Compare SUIT protocols
- SUIT-006 AmR mt D048 to investigate the dependence of H2O2 flux on the mt-membrane potantial on the N-control state in isolated mitochondria and tissue homogenate.
- SUIT-018 is a short protocol for oxygen dependence of O2 flux and H2O2 production on isolated mitochondria or tissue homogenate.
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 |
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 |
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 |
MitoPedia concepts:
SUIT protocol,
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MitoPedia methods:
Fluorometry