Department of Medical Cell Biology, Institute for Anatomy and Cell Biology, Heidelberg University.
Department of Neurogenetics, Max-Planck-Institute for Experimental Medicine.
J Vis Exp. 2021 Oct 20(176). doi: 10.3791/63073.
Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant thiol-disulfide redox buffer glutathione is considered a central player in antioxidant defenses. Therefore, measuring the mitochondrial glutathione redox potential provides useful information about mitochondrial redox status and oxidative stress. Glutaredoxin1-roGFP2 (Grx1-roGFP2) is a genetically encoded, green fluorescent protein (GFP)-based ratiometric indicator of the glutathione redox potential that has two redox-state-sensitive excitation peaks at 400 nm and 490 nm with a single emission peak at 510 nm. This article describes how to perform confocal live microscopy of mitochondria-targeted Grx1-roGFP2 in primary hippocampal and cortical neurons. It describes how to assess steady-state mitochondrial glutathione redox potential (e.g., to compare disease states or long-term treatments) and how to measure redox changes upon acute treatments (using the excitotoxic drug N-methyl-D-aspartate (NMDA) as an example). In addition, the article presents co-imaging of Grx1-roGFP2 and the mitochondrial membrane potential indicator, tetramethylrhodamine, ethyl ester (TMRE), to demonstrate how Grx1-roGPF2 can be multiplexed with additional indicators for multiparametric analyses. This protocol provides a detailed description of how to (i) optimize confocal laser scanning microscope settings, (ii) apply drugs for stimulation followed by sensor calibration with diamide and dithiothreitol, and (iii) analyze data with ImageJ/FIJI.
线粒体氧化还原稳态对于神经元的存活和功能至关重要。尽管线粒体含有多个氧化还原系统，但丰富的硫醇-二硫键氧化还原缓冲剂谷胱甘肽被认为是抗氧化防御的核心。因此，测量线粒体谷胱甘肽氧化还原电势提供了有关线粒体氧化还原状态和氧化应激的有用信息。谷氧还蛋白 1-roGFP2（Grx1-roGFP2）是一种遗传编码的、基于绿色荧光蛋白（GFP）的谷胱甘肽氧化还原电势的比率型指示剂，它在 400nm 和 490nm 处具有两个氧化还原状态敏感的激发峰，在 510nm 处具有单个发射峰。本文描述了如何在原代海马和皮质神经元中进行线粒体靶向 Grx1-roGFP2 的共聚焦活细胞显微镜观察。它描述了如何评估稳态线粒体谷胱甘肽氧化还原电势（例如，比较疾病状态或长期治疗），以及如何在急性处理时测量氧化还原变化（以兴奋性毒性药物 N-甲基-D-天冬氨酸（NMDA）为例）。此外，本文还介绍了 Grx1-roGFP2 与线粒体膜电位指示剂四甲基罗丹明乙酯（TMRE）的共成像，以展示 Grx1-roGPF2 如何与其他指示剂进行复用，用于多参数分析。本方案详细描述了如何（i）优化共聚焦激光扫描显微镜设置，（ii）应用药物进行刺激，然后用二硫苏糖醇（DTT）和二硫苏糖醇（DTT）进行传感器校准，以及（iii）使用 ImageJ/FIJI 分析数据。
