Glutathione (GSH) is a major antioxidant in cells, and plays vital roles in the cellular defense against oxidants and
in the regulation of redox signals. In a previous report, we demonstrated that stem cell function is critically affected
by heterogeneity and dynamic changes in cellular GSH concentration. Here, we present a detailed protocol for the
monitoring of GSH concentration in living stem cells using FreSHtracer, a real-time GSH probe. We describe the
steps involved in monitoring GSH concentration in single living stem cells using confocal microscopy and flow
cytometry. These methods are simple, rapid, and quantitative, and able to demonstrate intracellular GSH concentration
changes in real time. We also describe the application of FreSHtracer to the sorting of stem cells according to their
GSH content using flow cytometry. Typically, microscopic or flow cytometric analyses of FreSHtracer and
MitoFreSHtracer signals in living stem cells take ∼2∼3 h, and the fractionation of stem cells into subpopulations
on the basis of cellular GSH levels takes 3∼4.5 h. This method could be applied to almost every kind of mammalian
cell with minor modifications to the protocol described here.
Keywords: Glutathione, Real-time monitoring, Fluorescent probes, Stem cells