Visualization of Intracellular Hydrogen Peroxide with the Genetically Encoded Fluorescent Probe HyPer in NIH-3T3 Cells.

Autor: Ermakova YG; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.; European Molecular Biology Laboratory, Heidelberg, Germany., Mishina NM; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia., Schultz C; European Molecular Biology Laboratory, Heidelberg, Germany.; Oregon Health and Science University, Portland, OR, USA., Belousov VV; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia. belousov@ibch.ru.; Pirogov Russian National Research Medical University, Moscow, Russia. belousov@ibch.ru.; Georg August University of Göttingen, Göttingen, Germany. belousov@ibch.ru.
Jazyk: angličtina
Zdroj: Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2019; Vol. 1982, pp. 259-274.
DOI: 10.1007/978-1-4939-9424-3_15
Abstrakt: Reactive oxygen species (ROS) are involved in regulating normal physiological cell functions as second messengers as well as nonspecific damage of biomolecules in a pathological process known as oxidative stress. The HyPer family of genetically encoded probes are a useful noninvasive tool for monitoring the real-time dynamics of ROS in individual cells or model organisms. HyPer, the first genetically encoded probe for detection of hydrogen peroxide (H 2 O 2 ), is oxidized with high specificity and sensitivity by H 2 O 2 , leading to ratiometric changes in the fluorescence excitation spectrum of the probe. These changes can be detected with a wide range of commercial confocal and wide-field microscope systems. Here we describe a detailed protocol for ratiometric monitoring of H 2 O 2 produced by D-amino acid oxidase (DAAO) or by NADPH oxidase (NOX) in NIH-3T3 cells using the HyPer probe.
Databáze: MEDLINE
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