Int J Stem Cells.  2019 Jul;12(2):367-379. 10.15283/ijsc18151.

Monitoring Glutathione Dynamics and Heterogeneity in Living Stem Cells

• Jeong EM
• Shin JW
• Lim J
• Kim JH
• Kang H
• Yin Y
• Kim HM
• Kim Y
• Kim SG
• Kang HS
• Shin DM
• Choi K
• Kim IG

• KMID: 2465906
• DOI: http://doi.org/10.15283/ijsc18151

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.

Keyword

Glutathione; Real-time monitoring; Fluorescent probes; Stem cells

MeSH Terms

Flow Cytometry
Fluorescent Dyes
Glutathione*
Methods
Microscopy, Confocal
Oxidants
Oxidation-Reduction
Population Characteristics*
Stem Cells*
Fluorescent Dyes
Glutathione
Oxidants

Figure

• 

  Fig. 1 FreSHtracer, a reversible GSH probe. (a) The structure of the FreSHtracer backbone and its fluorescence spectral changes when it reacts with GSH. (b) The structures of R for FreSHtracer and MitoFreSHtracer.

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  Fig. 2 Imaging and monitoring of GSH concentration in single living cells. (a) Overview of the fluorescence confocal microscopy setup. (b, c) (i) Scheme for the whole-cell (b) and mitochondrial (c) staining of GSH. (ii, iii) sequential pseudo-color images with red hot scale (ii) and kinetics (iii) of F510/F580 ratio (FR) for FreSHtracer- or MitoFreSHtracer-loaded hUC-MSCs, which were treated with 0.5 mM diamide (DA) or 0.5 mM dithiothreitol (DTT).

• 

  Fig. 3 Flow cytometric analysis of living cells according to their GSH content. (a) Flowchart showing an overview of the procedure for measuring cellular GSH concentration using flow cytometry. (b, c) Flow cytometric analysis of GSH concentration in hUC-MSCs treated with 5 mM diamide (DA), 5 mM dithiothreitol (DTT), or 5 mM N-ethylmaleimide (NEM), using FreSHtracer (b; n=3 independent biological replicates) or MitoFreSHtracer (c; n=3 independent biological replicates).

• 

  Fig. 4 Flow cytometric sorting of living cells according to their GSH content. (a) Flowchart showing an overview of the procedure for the division of cells into subpopulations on the basis of their GSH content, using flow cytometry. (b) In vitro GSH assay of lysates and (c) count of colony-forming units of fibroblasts (CFU-F; n=3 independent biological replicates) in hUC-MSCs, sorted on the basis of FR.

Reference

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