活细胞中过氧化氢的局部生成与成像

Local Generation and Imaging of Hydrogen Peroxide in Living Cells.

作者信息

Bogdanova Yulia A, Schultz Carsten, Belousov Vsevolod V

机构信息

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia.

EMBL Heidelberg, Heidelberg, Germany.

出版信息

Curr Protoc Chem Biol. 2017 Jun 19;9(2):117-127. doi: 10.1002/cpch.20.

DOI:10.1002/cpch.20

PMID:28628200

Abstract

Described here is a localized H O generation-detection system consisting of a yeast D-amino acid oxidase (DAAO) and two spectrally distinct variants of biosensor, HyPer2 and HyPerRed based on circularly permutated yellow and red fluorescent proteins, respectively, which enables spatiotemporal production and examination of the intracellular H O dynamics. The protocol describes using this system in a simple cell culture model. We provide detailed instructions on imaging of H O generated by the activated DAAO. The system can be easily optimized for various combinations of cell types, conditions and DAAO/sensor subcellular localizations. © 2017 by John Wiley & Sons, Inc.

摘要

本文描述了一种局部过氧化氢生成检测系统，该系统由酵母D-氨基酸氧化酶（DAAO）和两种光谱不同的生物传感器变体组成，分别是基于环状排列的黄色和红色荧光蛋白的HyPer2和HyPerRed，它能够对细胞内过氧化氢动力学进行时空生成和检测。该方案描述了在简单细胞培养模型中使用此系统的方法。我们提供了关于活化DAAO产生的过氧化氢成像的详细说明。该系统可针对细胞类型、条件和DAAO/传感器亚细胞定位的各种组合轻松进行优化。© 2017约翰威立国际出版公司。