Detection of Intracellular Glutathione Using ThiolTracker Violet Stain and Fluorescence Microscopy

Bhaskar S. Mandavilli1, Michael S. Janes1

1 Invitrogen Corporation, Molecular Probes Labeling and Detection Technologies, Eugene, Oregon
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.35
DOI:  10.1002/0471142956.cy0935s53
Online Posting Date:  July, 2010
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Abstract

Glutathione plays an important role in protecting mammalian cells from oxidative stress and cell death. Because reduced glutathione (GSH) represents the large majority of intracellular free thiols, cell‐permeant, thiol‐reactive fluorescent probes represent potentially useful indicators of intracellular GSH. The ThiolTracker Violet stain (a registered trademark of Invitrogen) is a bright fluorescent probe that is highly reactive to thiols and can be used as a convenient and effective indicator of intracellular GSH and general redox status by a variety of detection modalities. While this probe has been validated in flow cytometry and microplate fluorimetry assays, the following method will describe details on the use of the ThiolTracker Violet dye in traditional fluorescence microscopy, as well as high‐content imaging and analysis. Curr. Protoc. Cytom. 53:9.35.1‐9.35.8. © 2010 by John Wiley & Sons, Inc.

Keywords: glutathione; ThiolTracker Violet; oxidative stress; cell based assays; high content imaging; fluorescence microscopy

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Mammalian cells of interest growing in culture, and appropriate incubation medium
  • Test compounds of interest
  • Dulbecco's phosphate‐buffered saline with Ca2+, Mg2+, glucose, and sodium pyruvate (DPBS, Invitrogen, cat. no. 14287‐080)
  • 20 µM ThiolTracker Violet dye working solution (see recipe)
  • 250× HCS NuclearMask Deep Red stain (Invitrogen, cat no. H10325; store desiccated at −20°C up to 1 year; also see recipe in Reagents and Solutions)
  • Dulbecco's phosphate buffered saline without Ca2+ and Mg2+ (DPBS, Invitrogen, cat. no. 14190‐144)
  • ProLong Gold antifade reagent (Invitrogen, cat. no. P36930; stable for 6 months when stored at −20°C)
  • Coverslips or 96‐well plates, appropriately coated for cell model
  • Cellomics ArrayScan VTI imager (Thermo Scientific; optional, for automated high‐content imaging and analysis)
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Figures

  •   FigureFigure 9.35.1 U‐2 OS cells were plated on Greiner 96‐well plates at a density of 10,000 cells per well and incubated overnight at 37°C. Cells were stained with increasing concentrations of ThiolTracker Violet dye and 1× HCS NuclearMask Deep Red stain for 30 min in thiol‐free DPBS without Ca2+ or Mg2+. The cells were washed once with the buffer and imaged on a Thermo Scientific Cellomics ArrayScan VTI. Fluorescence was plotted versus dye concentration. Optimal staining was achieved in this model using ThiolTracker Violet dye stain at 20 µM.
  •   FigureFigure 9.35.2 U‐2 OS cells were plated on Greiner 96‐well plates at a density of 10,000 cells per well and incubated overnight at 37°C. Cells were stained with 20 µM ThiolTracker Violet dye for various time points from 0 to 60 min in thiol‐free DPBS without Ca2+ or Mg2+. The cells were washed once with PBS and counterstained with 1× HCS NuclearMask Deep Red stain and imaged on a Thermo Scientific Cellomics ArrayScan VTI. In this figure, fluorescence of cells stained with 20 µM ThiolTracker Violet dye was plotted against time. Satisfactory staining with 20 µM ThiolTracker Violet dye was achieved in this model using a 30‐min incubation period.
  •   FigureFigure 9.35.3 HepG2 cells plated on collagen‐coated 96‐well plates and treated with various doses (2‐fold dilutions) of buthionine sulfoximine (BSO, 4 mM highest dose) and diethyl maleate (DEM, 2 mM highest dose) in combination at 2:1 molar ratios for 2 hr before staining with 20 µM ThiolTracker Violet stain and 1× HCS NuclearMask Deep Red stain for 30 min in thiol‐free DPBS without Ca2+ or Mg2+. (A) The cells were washed once with Ca2+ and Mg2+–free DPBS and imaged on a Thermo Scientific Cellomics ArrayScan VTI. (B) The fluorescence intensity of ThiolTracker Violet stain in the cytoplasmic regions was plotted against various concentrations of BSO and DEM. The combined EC50 value of BSO and DEM is 196 µM.
  •   FigureFigure 9.35.4 Fluorescence absorption and emission spectra for ThiolTracker Violet dye obtained by scanning probe‐GSH conjugates in Ca2+ and Mg2+–free DPBS (pH 7.4).

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Literature Cited

Literature Cited
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