Difference between revisions of "SUIT-009 AmR ce-pce D019"
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{{MitoPedia | {{MitoPedia | ||
|abbr= | |abbr=H<sub>2</sub>O<sub>2</sub> RET ce-pce S_L | ||
|description=[[File:1S;2D;3P;4Rot | |description=[[File:Ce1;1Dig;1S;2D;3P;4Rot;5Ama.png|520px|SUIT9]] | ||
|info=''' | |info='''B''': short protocol for H<sub>2</sub>O<sub>2</sub> (AmR) in ce: non-permeabilized cells, pce: permeabilized cells -'''[[SUIT-009]]''' | ||
|SUIT number=D019_ce1;1Dig;1S;2D;3P;4Rot;5Ama | |SUIT number=D019_ce1;1Dig;1S;2D;3P;4Rot;5Ama | ||
|O2k-Application: AmR | |O2k-Application: AmR | ||
}} | }} | ||
SUIT-009 is a short protocol for simultaneous determination of O<sub>2</sub> and [[MiPNet20.14 AmplexRed H2O2-production |H<sub>2</sub>O<sub>2</sub>]] flux in [[permeabilized cells]]. Living cells are added first to the O2k chamber and then permeabilized as a part of the protocol. Succinate (S) supports [[reverse electron flow from CII to CI]] (RET) with high H<sub>2</sub>O<sub>2</sub> production in the LEAK state at low O<sub>2</sub> flow, S<sub>''L''</sub>. Respiration is stimulated by ADP (OXPHOS capacity, S<sub>''P''</sub>), while H<sub>2</sub>O<sub>2</sub> flux declines dramatically. Pyruvate (P) supports the [[NADH Electron transfer-pathway state|NADH-pathway]] and thus the convergent NS-pathway with little further effect on H<sub>2</sub>O<sub>2</sub> flux, NS<sub>''P''</sub>). Malate is not required, since oxaloacetate is formed from succinate-fumarate-malate. Antimycin A (Ama) inhibits Complex III (CIII) at the Q<sub>o</sub> level inhibiting respiration to the level of ''Rox'', and generally increases the H<sub>2</sub>O<sub>2</sub> flux. The sensitivity of the Amplex UltraRed® (AmR) changes over experimental time and upon addition of chemicals. To correct H<sub>2</sub>O<sub>2</sub> flux for the sensitivity changes, several [[MiPNet20.14_AmplexRed_H2O2-production#Calibration_with_H2O2|H<sub>2</sub>O<sub>2</sub> calibration steps]] are done during the experiment. The experimental control for O<sub>2</sub> flux in the absence AmR is carried out with [[SUIT-009 O2 ce-pce D016]]. | |||
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 µM 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__ | ||
Communicated by [[Huete-Ortega M]], [[Cardoso LHD]], [[Doerrier C]], [[Komlodi T]], [[Gnaiger E]] (last update 2019-09-17) | |||
{{Template:SUIT-009 AmR pce D019}} | |||
== | == Strengths and limitations == | ||
::::* [[SUIT-009 AmR mt | :::* Information is obtained on respiration and ROS production in the S- and NS-pathway. | ||
:::: | :::* [[SUIT-009 O2 ce-pce D016]] determination of O<sub>2</sub> flux on permeabilized cells as a control for SUIT-009 AmR ce-pce D019 to test the inhibitory effect of the fluorescence dye on the mt-respiration. | ||
:::* 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 should be done in parallel. | |||
:::: | ::: + This protocol is designed to evaluate RET in the S-linked pathway in the LEAK state, which is decreased by ADP due to reduction of the protonmotive force, ''pmF''. | ||
::: + Short duration of the experiment. | |||
::: - CIV activity and cytochrome ''c'' test cannot be performed together with the fluorescence assay. | |||
::: - The H<sub>2</sub>O<sub>2</sub> flux will be much lower compared to that observed with isolated mitochondria, which might be explained by the high antioxidant capacity of cytosolic compounds. | |||
::: - In the case of HEK293T cells, however, due to the high malate-linked anaplerotic capacity, malate is converted not only to oxaloacetate but also to pyruvate, such that addition of succinate alone supports NS-pathway capacity (no effect of pyruvate on OXPHOS capacity. Under these conditions, reverse electron transfer is inhibited in the LEAK state, which prevents excessively high H<sub>2</sub>O<sub>2</sub> flow. | |||
== | |||
== Compare SUIT protocols == | |||
:::* [[SUIT-006 AmR mt D048]] to investigate the dependence of H<sub>2</sub>O<sub>2</sub> flux on the mt-membrane potential in the N-control state in isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber). | |||
:::* [[SUIT-018]] is a short protocol to study the oxygen dependence of O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> production in isolated mitochondria or tissue homogenate (except of liver). | |||
== Chemicals and syringes == | |||
{{Template:Chemicals AmR}} | |||
{{Template:Chemicals ce}} | |||
{{Template:Chemicals SUIT-009}} | |||
::: Suggested stock concentrations are shown in the specific DL-Protocol. | |||
== References == | == References == | ||
{{#ask:[[Category:Publications]] [[Additional label::SUIT-009 AmR pce D019 | {{#ask:[[Category:Publications]] [[Additional label::SUIT-009 AmR ce-pce D019]] | ||
| ?Was published in year=Year | | ?Was published in year=Year | ||
| ?Has title=Reference | | ?Has title=Reference | ||
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}} | }} | ||
::: | {{#ask:[[Category:Abstracts]] [[Additional label::SUIT-009 AmR ce-pce D019]] [[Instrument and method::O2k-Protocol]] | ||
| ?Was submitted in year=Year | |||
| ?Has title=Reference | |||
| ?Mammal and model=Organism | |||
| ?Tissue and cell=Tissue;cell | |||
| format=broadtable | |||
| limit=5000 | |||
| offset=0 | |||
| sort=Was submitted in year | |||
| order=descending | |||
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{{MitoPedia concepts | {{MitoPedia concepts | ||
|mitopedia concept=SUIT protocol, SUIT | |mitopedia concept=SUIT protocol, SUIT B, Find | ||
}} | }} | ||
{{MitoPedia methods | {{MitoPedia methods | ||
|mitopedia method=Fluorometry | |mitopedia method=Fluorometry | ||
}} | }} |
Latest revision as of 00:10, 13 April 2021
Description
Abbreviation: H2O2 RET ce-pce S_L
Reference: B: short protocol for H2O2 (AmR) in ce: non-permeabilized cells, pce: permeabilized cells -SUIT-009
SUIT number: D019_ce1;1Dig;1S;2D;3P;4Rot;5Ama
SUIT-009 is a short protocol for simultaneous determination of O2 and H2O2 flux in permeabilized cells. Living cells are added first to the O2k chamber and then permeabilized as a part of the protocol. Succinate (S) supports reverse electron flow from CII to CI (RET) with high H2O2 production in the LEAK state at low O2 flow, SL. Respiration is stimulated by ADP (OXPHOS capacity, SP), while H2O2 flux declines dramatically. Pyruvate (P) supports the NADH-pathway and thus the convergent NS-pathway with little further effect on H2O2 flux, NSP). Malate is not required, since oxaloacetate is formed from succinate-fumarate-malate. Antimycin A (Ama) inhibits Complex III (CIII) at the Qo level inhibiting respiration to the level of Rox, and generally increases the H2O2 flux. The sensitivity of the Amplex UltraRed® (AmR) changes over 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 experimental control for O2 flux in the absence AmR is carried out with SUIT-009 O2 ce-pce D016.
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 µM O2. SUIT-018 AmR mt D041 has been designed to study O2 dependence of H2O2 flux in mitochondrial preparations.
Communicated by Huete-Ortega M, Cardoso LHD, Doerrier C, Komlodi T, Gnaiger E (last update 2019-09-17)
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
- Information is obtained on respiration and ROS production in the S- and NS-pathway.
- SUIT-009 O2 ce-pce D016 determination of O2 flux on permeabilized cells as a control for SUIT-009 AmR ce-pce D019 to test the inhibitory effect of the fluorescence dye on the mt-respiration.
- 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 should be done in parallel.
- + This protocol is designed to evaluate RET in the S-linked pathway in the LEAK state, which is decreased by ADP due to reduction of the protonmotive force, pmF.
- + Short 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 cytosolic compounds.
- - In the case of HEK293T cells, however, due to the high malate-linked anaplerotic capacity, malate is converted not only to oxaloacetate but also to pyruvate, such that addition of succinate alone supports NS-pathway capacity (no effect of pyruvate on OXPHOS capacity. Under these conditions, reverse electron transfer is inhibited in the LEAK state, which prevents excessively high H2O2 flow.
Compare SUIT protocols
- SUIT-006 AmR mt D048 to investigate the dependence of H2O2 flux on the mt-membrane potential in the N-control state in isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber).
- SUIT-018 is a short protocol to study the oxygen dependence of O2 flux and H2O2 production in isolated mitochondria or tissue homogenate (except of liver).
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 |
---|---|---|
1Dig | Digitonin (Dig) |
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1S | Succinate (S) | |
2D | ADP (D) | |
3P | Pyruvate (P) | |
4Rot | Rotenone (Rot) | |
5Ama | Antimycin A (Ama) |
- Suggested stock concentrations are shown in the specific DL-Protocol.
References
MitoPedia concepts: SUIT protocol, SUIT B, Find
MitoPedia methods:
Fluorometry