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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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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Basic Protocol 1:

  • 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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Literature Cited

Literature Cited
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