SUIT-009 AmR mt D021: Difference between revisions
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::::* [[SUIT-009 AmR mt D021]] | ::::* [[SUIT-009 AmR mt D021]] | ||
::::* [[SUIT-009 AmR pce D019]] | ::::* [[SUIT-009 AmR pce D019]] | ||
::::* [[SUIT-009 O2 mt | ::::* [[SUIT-009 O2 mt D015]] | ||
::::* [[SUIT-009 O2 pce D16]] | ::::* [[SUIT-009 O2 pce D16]] | ||
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== 1S;2D;3P;4Rot;5Ama-== | == 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. | :::: 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. | ||
:::: For O2-Application, choose SUIT-009 O2 mt | :::: For O2-Application, choose [[SUIT-009 O2 mt D015]]; for O2 and H<sub>2</sub>O<sub>2</sub> measurements (AmR), choose: D021_1S;2D;3P;4Rot;5Ama. | ||
Revision as of 11:50, 17 January 2019
- high-resolution terminology - matching measurements at high-resolution
SUIT-009 AmR mt D021
Description
Abbreviation: NS(P)
Reference: A: SUIT-009 short protocol for H2O2 (AmR) in mitochondrial preparations (isolated mitochondria and tissue homogenate).
SUIT number: D021_1S;2D;3P;4Rot;5Ama
O2k-Application: AmR
MitoPedia concepts:
SUIT protocol,
SUIT A,
Find
MitoPedia methods:
Fluorometry
- SUIT-category: NS(P)
- SUIT protocol pattern: orthogonal 1S;2D;3P;4Rot
SUIT protocols
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.
- For O2-Application, choose SUIT-009 O2 mt D015; for O2 and H2O2 measurements (AmR), choose: D021_1S;2D;3P;4Rot;5Ama.
