利用基因编码蛋白传感器和荧光染料测量钙和活性氧。

Measuring Calcium and ROS by Genetically Encoded Protein Sensors and Fluorescent Dyes.

作者信息

Gibhardt Christine S, Vultur Adina, Bogeski Ivan

机构信息

Molecular Physiology, Institute of Cardiovascular Physiology, University Medical Center, Georg-August-University, Göttingen, Germany.

出版信息

Methods Mol Biol. 2019;1925:183-196. doi: 10.1007/978-1-4939-9018-4_17.

DOI:10.1007/978-1-4939-9018-4_17

PMID:30674028

Abstract

Oxidative modifications of cellular building blocks such as proteins, lipids, and DNA have a major impact on cell behavior, fate, and clinical outcome. Reactive oxygen species (ROS) are important factors that influence these redox processes. Calcium ion (Ca) dynamics and signals are also essential regulators of key cellular processes. Therefore, the combined and precise monitoring of ROS and Ca in single cells, with a high spatial and temporal resolution and in physiological environments, is essential to better understand their functional impact. Here, we describe protocols to detect one of the most prominent ROS (hydrogen peroxide, HO) using genetically encoded protein sensors and fluorescent dyes. We also provide guidelines on how to simultaneously detect Ca and HO and how to examine the influence of Ca signals on cellular ROS production and vice versa.

摘要

细胞组成成分如蛋白质、脂质和DNA的氧化修饰对细胞行为、命运及临床结果有重大影响。活性氧（ROS）是影响这些氧化还原过程的重要因素。钙离子（Ca）动态变化和信号也是关键细胞过程的重要调节因子。因此，在生理环境中以高时空分辨率对单细胞中的ROS和Ca进行联合精确监测，对于更好地理解它们的功能影响至关重要。在此，我们描述了使用基因编码蛋白传感器和荧光染料检测最主要的ROS之一（过氧化氢，H₂O₂）的实验方案。我们还提供了关于如何同时检测Ca和H₂O₂以及如何研究Ca信号对细胞ROS产生的影响，反之亦然的指导原则。