SUIT-006 AmR mt D048: Difference between revisions
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{{MitoPedia | {{MitoPedia | ||
|abbr= | |abbr=CCP mt PM - AmR | ||
|description=[[File:1PM;2D;3Omy;4U;5Ama.png| | |description=[[File:1PM;2D;3Omy;4U;5Ama.png|400px]] | ||
|info='''A''': | |info='''A''': short protocol for simultaneous determination of O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)- '''[[SUIT-006]]''' | ||
|application=AmR | |application=AmR | ||
|SUIT number=D048_1PM;2D;3Omy;4U;5Ama | |SUIT number=D048_1PM;2D;3Omy;4U;5Ama | ||
}} | }} | ||
SUIT-006 AmR mt D048 is a short protocol to study simultaneously the O<sub>2</sub> flux and [[Hydrogen peroxide|H<sub>2</sub>O<sub>2</sub>]] flux (with the fluorescent dye [[Amplex UltraRed]]), in particular to study the dependence of H<sub>2</sub>O<sub>2</sub> flux on the [[mt-membrane potential]]. In this protocol , the [[NADH Electron transfer-pathway state]] can be assessed in [[Mitochondrial preparations| mitochondrial preparations]] such as isolated mitochondria, tissue homogenates (except of liver homogenate) and permeabilized cells (already permeabilized when they are added to the chamber) in a wide variety of species and tissues. After addition of the NADH-linked substrates, the addition of ADP depolarises the mt-membrane which leads to the decrease of the H<sub>2</sub>O<sub>2</sub> flux. ย | |||
[[Oligomycin]] (Omy) is used to induce a LEAK state of respiration via the inhibition of the ATP synthase. Omy hyperpolarises the mt-membrane resulting in increased H<sub>2</sub>O<sub>2</sub> production, while the addition of the uncoupler decreases the H<sub>2</sub>O<sub>2</sub> generation owing to the depolarisation of the mt-membrane. | |||
Since higher concentrations of Omy can decrease the ET state induced upon addition of uncoupler, the optimal concentration of Omy has to be determined. ย | |||
According to our results, the H<sub>2</sub>O<sub>2</sub> production is linearly dependent on the O<sub>2</sub> concentration in MiR05-Kit, therefore, during the measurements the O<sub>2</sub> concentration has to be well-defined. In this protocol, we suggest not to go under ~100 ยตM O<sub>2</sub>. [[SUIT-018 AmR mt D041]] has been designed to study O<sub>2</sub> dependence of H<sub>2</sub>O<sub>2</sub> flux in mitochondrial preparations. | |||
__TOC__ | __TOC__ | ||
ย Communicated byย [[Komlodi T]] (last update 2019- | ย Communicated byย [[Komlodi T]], [[Cardoso LHD]], [[Gnaiger E]] (last update 2019-07-05) ย | ||
== Representative traces == | |||
[[File:SUIT-006 AmR mt D048 O2.png|600px]] | |||
[[File:SUIT-006 AmR mt D048.png|600px]] | |||
{{Template:SUIT-006 AmR mt D048}} | {{Template:SUIT-006 AmR mt D048}} | ||
== Strengths and limitations == | == Strengths and limitations == | ||
:::* This protocol can be | :::* This protocol can be runned under low oxygen concentration to test the oxygen dependence of mt-ROS production. For more technical details see: [[SUIT-018 AmR mt D031]], [[SUIT-018 AmR mt D041]]. | ||
:::* To study the inhibitory effect of the AmR assay on the respiration using living cells, the following protocols are recommended: [[SUIT 003 AmR ce D058]] with AmR and [[SUIT 003 AmR ce D059]] with carrier titration as a control. These two protocols have to be done in parallel. ย | |||
:::+ Reasonable duration of the experiment. | :::+ Reasonable duration of the experiment. | ||
:::- CIV activity cannot be determined and | :::- CIV activity cannot be determined and cytochrome ''c'' test cannot be performed together with the fluorescence dyes. | ||
:::+ This protocol can be extended with the Complex IV module in the following protocols: [[SUIT-006 O2 mt D047]]. ย | :::+ This protocol can be extended with the Complex IV module in the following protocols: [[SUIT-006 O2 mt D047]]. ย | ||
:::+ Cytochrome ''c'' test can be performed in the following protocols:[[SUIT-006 O2 mt D047]]. ย | :::+ Cytochrome ''c'' test can be performed in the following protocols:[[SUIT-006 O2 mt D047]]. ย | ||
:::- This protocol cannot be | :::- This protocol cannot be runned with liver homogenate. ย | ||
:::- Omy concentration has to be determined. Higher concentrations of Omy may inhibit the ET state. | :::- Omy concentration has to be determined. Higher concentrations of Omy may inhibit the ET state. | ||
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== Compare SUIT protocols == | == Compare SUIT protocols == | ||
== Chemicals and syringes == | |||
{{Template:Chemicals AmR}} | |||
{{Template:Chemicals SUIT-006 Fluo}} | |||
::: Suggested stock concentrations are shown in the specific DL-Protocol. | |||
== References == | == References == |
Latest revision as of 23:51, 12 April 2021
Description
Abbreviation: CCP mt PM - AmR
Reference: A: short protocol for simultaneous determination of O2 flux and H2O2 in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)- SUIT-006
SUIT number: D048_1PM;2D;3Omy;4U;5Ama
O2k-Application: AmR
SUIT-006 AmR mt D048 is a short protocol to study simultaneously the O2 flux and H2O2 flux (with the fluorescent dye Amplex UltraRed), in particular to study the dependence of H2O2 flux on the mt-membrane potential. In this protocol , the NADH Electron transfer-pathway state can be assessed in mitochondrial preparations such as isolated mitochondria, tissue homogenates (except of liver homogenate) and permeabilized cells (already permeabilized when they are added to the chamber) in a wide variety of species and tissues. After addition of the NADH-linked substrates, the addition of ADP depolarises the mt-membrane which leads to the decrease of the H2O2 flux. Oligomycin (Omy) is used to induce a LEAK state of respiration via the inhibition of the ATP synthase. Omy hyperpolarises the mt-membrane resulting in increased H2O2 production, while the addition of the uncoupler decreases the H2O2 generation owing to the depolarisation of the mt-membrane. Since higher concentrations of Omy can decrease the ET state induced upon addition of uncoupler, the optimal concentration of Omy has to be determined.
According to our results, the H2O2 production is linearly dependent on the O2 concentration in MiR05-Kit, therefore, during the measurements the O2 concentration has to be well-defined. In this protocol, we suggest not to go under ~100 ยตM O2. SUIT-018 AmR mt D041 has been designed to study O2 dependence of H2O2 flux in mitochondrial preparations.
Communicated by Komlodi T, Cardoso LHD, Gnaiger E (last update 2019-07-05)
Representative traces
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) |
---|---|---|---|---|
1PM | PML(n) | 1PM
| ||
2D | PMP | 1PM;2D
| ||
3Omy | PML(Omy) | 1PM;2D;3Omy
| ||
4U | PME | 1PM;2D;3Omy;4U
| ||
5Ama | ROX | 1PM;2D;3Omy;4U;5Ama
|
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- Coupling control
- Pathway control
- ยป Electron transfer pathway
- ยป Fatty acid oxidation pathway control state, F
- ยป NADH electron transfer-pathway state, N
- ยป Succinate pathway control state, S
- ยป NS-pathway control state, NS
- ยป Glycerophosphate pathway control state, Gp
- ยป Complex IV single step, CIV
- ยป Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- ยป Glutamate, G
- ยป Glycerophosphate, Gp
- ยป Malate, M
- ยป Octanoylcarnitine, Oct
- ยป Pyruvate, P
- ยป Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
- This protocol can be runned under low oxygen concentration to test the oxygen dependence of mt-ROS production. For more technical details see: SUIT-018 AmR mt D031, SUIT-018 AmR mt D041.
- To study the inhibitory effect of the AmR assay on the respiration using living cells, the following protocols are recommended: SUIT 003 AmR ce D058 with AmR and SUIT 003 AmR ce D059 with carrier titration as a control. These two protocols have to be done in parallel.
- + Reasonable duration of the experiment.
- - CIV activity cannot be determined and cytochrome c test cannot be performed together with the fluorescence dyes.
- + This protocol can be extended with the Complex IV module in the following protocols: SUIT-006 O2 mt D047.
- + Cytochrome c test can be performed in the following protocols:SUIT-006 O2 mt D047.
- - This protocol cannot be runned with liver homogenate.
- - Omy concentration has to be determined. Higher concentrations of Omy may inhibit the ET state.
Compare SUIT protocols
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
H2O2 | Hydrogen Peroxide (H2O2) |
Step | Chemical(s) and link(s) | Comments |
---|---|---|
0DTPA | DTPA | This step can be skipped |
0SOD | Superoxide Dismutase (SOD) | |
0HRP | Horseradish peroxidase (HRP) | |
0AmR | Amplex UltraRed (AmR) |
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1PM | Pyruvate (P) and Malate (M) | |
2D | ADP (D) | |
3Omy | Oligomycin (Omy) | |
4U | Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) | Can be substituted for other uncoupler |
5Ama | Antimycin A (Ama) |
- Suggested stock concentrations are shown in the specific DL-Protocol.
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 |
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