Glutathione (GSH) is a chief cellular antioxidant, affecting stem cell functions. The cellular GSH level is dynamically altered by the redox buffering system and transcription factors, including NRF2. Additionally, GSH is differentially regulated in each organelle. We previously reported a protocol for monitoring the real-time GSH levels in live stem cells using the reversible GSH sensor FreSHtracer. However, GSH-based stem cell analysis needs be comprehensive and organelle-specific. Hence, in this study, we demonstrate a detailed protocol to measure the GSH regeneration capacity (GRC) in living stem cells by measuring the intensities of the FreSHtracer and the mitochondrial GSH sensor MitoFreSHtracer using a high-content screening confocal microscope. This protocol typically analyses the GRC in approximately 4 h following the seeding of the cells onto plates. This protocol is simple and quantitative. With some minor modifications, it can be employed flexibly to measure the GRC for the whole-cell area or just the mitochondria in all adherent mammalian stem cells.

中文翻译：

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测量干细胞中的谷胱甘肽再生能力。

谷胱甘肽 (GSH) 是一种主要的细胞抗氧化剂，影响干细胞功能。细胞 GSH 水平由氧化还原缓冲系统和转录因子（包括 NRF2）动态改变。此外，GSH 在每个细胞器中受到不同的调节。我们之前报道了一种使用可逆 GSH 传感器 FreSHtracer 监测活干细胞中实时 GSH 水平的方案。然而，基于 GSH 的干细胞分析需要全面且针对细胞器。因此，在这项研究中，我们展示了一个详细的方案，通过使用高内涵筛选共聚焦显微镜测量 FreSHtracer 和线粒体 GSH 传感器 MitoFreSHtracer 的强度来测量活干细胞中的 GSH 再生能力 (GRC)。该方案通常在将细胞接种到平板上后约 4 小时内分析 GRC。该协议简单且定量。经过一些细微的修改，它可以灵活地用于测量全细胞区域或所有贴壁哺乳动物干细胞中线粒体的 GRC。