Monitoring Glutathione Dynamics and Heterogeneity in Living Stem Cells
- Author:
Eui Man JEONG
1
;
Ji Woong SHIN
;
Jisun LIM
;
Ju Hwan KIM
;
Hyewon KANG
;
Yingfu YIN
;
Hye Mi KIM
;
YongHwan KIM
;
Sun Gi KIM
;
Heun Soo KANG
;
Dong Myung SHIN
;
Kihang CHOI
;
In Gyu KIM
Author Information
- Publication Type:Case Report
- Keywords: Glutathione; Real-time monitoring; Fluorescent probes; Stem cells
- MeSH: Flow Cytometry; Fluorescent Dyes; Glutathione; Methods; Microscopy, Confocal; Oxidants; Oxidation-Reduction; Population Characteristics; Stem Cells
- From:International Journal of Stem Cells 2019;12(2):367-379
- CountryRepublic of Korea
- Language:English
- Abstract: 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.
