Difference between revisions of "SUIT-009 AmR ce-pce D019"
From Bioblast
Line 1: | Line 1: | ||
{{MitoPedia | {{MitoPedia | ||
|abbr=NS(P) | |abbr=NS(P) | ||
|description=[[File: | |description=[[File:SUIT-009 O2 pce D019.png|400px|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 | ||
Line 8: | Line 8: | ||
::: '''[[Categories of SUIT protocols |SUIT-category]]:''' NS(P) | ::: '''[[Categories of SUIT protocols |SUIT-category]]:''' NS(P) | ||
::: '''[[SUIT protocol pattern]]:''' orthogonal 1S;2D;3P;4Rot;5Ama | ::: '''[[SUIT protocol pattern]]:''' orthogonal 1S;2D;3P;4Rot;5Ama | ||
:::SUIT 9 is a short protocol for simultaneous determination of O<sub>2</sub> flux and the rate of [[MiPNet20.14 AmplexRed H2O2-production|H<sub>2</sub>O<sub>2</sub> production]] (H<sub>2</sub>O<sub>2</sub> flux) on isolated mitochondria,tissue homogenate (except of liver) and permeabilized cells. Succinate (S) supports the reverse electron transfer (RET) initiated H<sub>2</sub>O<sub>2</sub> production in the LEAK state. Addition of ADP decreases H<sub>2</sub>O<sub>2</sub> flux due to depolarization of the mitochondrial membrane. Pyruvate (P) supports [[NADH Electron transfer-pathway state|NADH-pathway (N)]], and usually in the presence of ADP it does not increase further the H<sub>2</sub>O<sub>2</sub> flux. Antimycin A (Ama) inhibits Complex III (CIII) and results in the elevation of the H<sub>2</sub>O<sub>2</sub> flux. The sensitivity of the Amplex UltraRed® assay (for determining H<sub>2</sub>O<sub>2</sub> production) is changing over the 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 measurement is carried out in MiR05-Kit at 37 °C. | |||
__TOC__ | __TOC__ | ||
Communicated by [[Iglesias-Gonzalez J]], [[Komlodi T]] and [[Gnaiger E]] (last update 2019-01-22) | Communicated by [[Iglesias-Gonzalez J]], [[Komlodi T]] and [[Gnaiger E]] (last update 2019-01-22) | ||
== | == DLP applications == | ||
::::* [[SUIT-009 AmR mt D021]] | ::::* [[SUIT-009 AmR mt D021]] | ||
::::* [[SUIT-009 AmR pce D019]] | ::::* [[SUIT-009 AmR pce D019]] | ||
Line 18: | Line 20: | ||
[[File:SUIT-9 AmR pce D19 Overview.png|right|600px]] | [[File:SUIT-9 AmR pce D19 Overview.png|right|600px]] | ||
== References == | == References == | ||
Line 33: | Line 33: | ||
| order=descending | | order=descending | ||
}} | }} | ||
{{Template:SUIT-009}} | |||
== Strengths and limitations == | == Strengths and limitations == | ||
Line 39: | Line 41: | ||
::: - 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 the cytosol. | ::: - 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 the cytosol. | ||
::: - In some cell lines e.g. HEK293T, P does not have any effect because of the downregulation of the N-linked pathway. | ::: - 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 == | |||
{{MitoPedia concepts | {{MitoPedia concepts |
Revision as of 18:40, 22 January 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
- 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.
Communicated by Iglesias-Gonzalez J, Komlodi T and Gnaiger E (last update 2019-01-22)
DLP applications
References
Steps and respiratory states
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
|
Step | Respiratory state | Pathway control | ET-Complex | Comment |
---|---|---|---|---|
## AsTm | AsTmE | CIV | CIV | |
## Azd | CHB |
- 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
- + 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.
- - 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
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Fluorometry