Monitoring Glutathione Dynamics and Heterogeneity in Living Stem Cells
International Journal of Stem Cells
;
: 367-379, 2019.
Artigo
em Inglês
| WPRIM
| ID: wpr-764067
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.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Oxirredução
/
Células-Tronco
/
Características da População
/
Oxidantes
/
Microscopia Confocal
/
Citometria de Fluxo
/
Corantes Fluorescentes
/
Glutationa
/
Métodos
Idioma:
Inglês
Revista:
International Journal of Stem Cells
Ano de publicação:
2019
Tipo de documento:
Artigo
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