Measuring Glutathione Regeneration Capacity in Stem Cells

Int J Stem Cells. 2023 Aug 30;16(3):356-362. doi: 10.15283/ijsc23047. Epub 2023 Jun 30.


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.

Keywords: FreSHtracer; Glutathione; Glutathione regeneration capacity; Stem cells.

Grants and funding

Acknowledgments We thank all Cell2in members for their assistance with this study. This work was supported by a Jeju National University research grant in 2021.