Difference between revisions of "SUIT-018"
Line 28: | Line 28: | ||
== Strengths and limitations == | == Strengths and limitations == | ||
:::* Many fluorescence dyes can inhibit components of the ET system and thus, affecting the respiration. Therefore, a control run of the protocol should be done in the absence of the fluorescence dye. The following protocols can be used: SUIT-0018 O2 mt D0'##. | |||
:::+ Reasonable duration of the experiment. | :::+ Reasonable duration of the experiment. | ||
:::+ Simple protocol to evaluate the oxygen dependence of H<sub>2</sub>O<sub>2</sub> production in LEAK and OXPHOS state. | :::+ Simple protocol to evaluate the oxygen dependence of H<sub>2</sub>O<sub>2</sub> production in LEAK and OXPHOS state. |
Revision as of 12:06, 4 March 2019
Description
Abbreviation: NS(GM)
Reference: A: Short protocol for oxygen dependence of O2 flux and H2O2 production on isolated mitochondria or tissue homogenate »Versions
- SUIT-category:NS(GM)
- SUIT protocol pattern: linear 1GMS;2D;-
SUIT-018 is a short protocol to study simultaneously the oxygen dependence of O2 flux and H2O2 production on isolated mitochondria or tissue homogenate (except of liver homogenate). Is it possible to run the protocol under different oxygen concentration conditions: 1.) under tissue normoxia (50-60 µM ~ [O2]) SUIT-018 AmR mt D031, 2.) under tissue hyperoxia (> ~120 µM = [O2]) SUIT-018 AmR mt D040 or 3.) modifying the oxygen concentration during the experiment SUIT-018 AmR mt D041. The oxygen concentration in the chamber can be altered injecting nitrogen with a syringe. It is recommended to run both SUIT-018 AmR mt D031 and SUIT-018 AmR mt D040 in parallel or SUIT-018 AmR mt D041 alone in order to test the H2O2 production at different oxygen concentrations.
Communicated by Komlodi T and Iglesias-Gonzalez J(last update 2019-02-12)
Specific SUIT protocols
- SUIT-018 AmR mt D031 simultaneous determination of O2 and H2O2 flux on isolated mitochondria and tissue homogenate at low oxygen concentration (tissue normoxia).
- SUIT-018 AmR mt D040 simultaneous determination of O2 and H2O2 flux on isolated mitochondria and tissue homogenate at high oxygen concentration (tissue hyperoxia).
- SUIT-018 AmR mt D041 simultaneous determination of O2 and H2O2 flux on isolated mitochondria and tissue homogenate changing the oxygen concentration in the same protocol.
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) |
---|---|---|---|---|
1GMS | GMSL(n) | NS | CI&CII | 1GMS
|
2D | GMSP | NS | CI&CII | 1GMS;2D
|
3Ama | ROX | 1GMS;2D;3Ama
|
Strengths and limitations
- Many fluorescence dyes can inhibit components of the ET system and thus, affecting the respiration. Therefore, a control run of the protocol should be done in the absence of the fluorescence dye. The following protocols can be used: SUIT-0018 O2 mt D0'##.
- + Reasonable duration of the experiment.
- + Simple protocol to evaluate the oxygen dependence of H2O2 production in LEAK and OXPHOS state.
- - Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate.
- - Reoxygenation and nitrogen injection could lead to bubble formation in the O2k-Chamber.
- - CIV activity and cytochrome c test cannot be performed together with the fluorescence.
Compare SUIT protocols
- SUIT-006 AmR mt D048 to investigate the dependence of H2O2 flux on the mt-membrane potential on the N-control state in isolated mitochondria and tissue homogenate.
- SUIT-009 to investigate H2O2 production driven by the reverse electron transfer (RET) on isolated mitochondria, tissue homogenate and permeabilized 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 | 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 |
MitoPedia concepts: MiP concept, SUIT protocol, Recommended
MitoPedia methods:
Fluorometry