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Related Experiment Videos

Evaluation of methods for measuring cellular glutathione content using flow cytometry

D W Hedley1, S Chow

  • 1Department of Pathology, Ontario Cancer Institute/Princess Margaret Hospital, Toronto, Canada.

Cytometry
|April 1, 1994
PubMed
Summary

Monobromobimane is the best stain for measuring cellular glutathione (GSH) in human cells using flow cytometry, offering reproducible staining. Other stains like monochlorobimane and mercury orange have limitations with human cells or background fluorescence.

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Area of Science:

  • Cellular Biology
  • Biochemistry
  • Analytical Chemistry

Background:

  • Flow cytometry is a key technique for analyzing cellular components.
  • Accurate measurement of cellular glutathione (GSH) is crucial for understanding cellular health and disease.
  • Existing flow cytometric stains for GSH have variable performance across different cell types.

Purpose of the Study:

  • To evaluate currently available flow cytometric stains for cellular glutathione (GSH) detection.
  • To compare the efficacy and specificity of various GSH stains in human and rodent cell lines.
  • To identify the optimal stain for reproducible GSH measurement in human cells via flow cytometry.

Main Methods:

  • Evaluation of monochlorobimane, monobromobimane, mercury orange, o-phthaldialdehyde, and chloromethyl fluorescein.

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  • Testing stains on human and rodent cell lines under varying concentrations, incubation times, and temperatures.
  • Utilizing GSH-depleted cells to assess linearity and background fluorescence.

    • Main Results:

    • Monochlorobimane showed high specificity for rodent GSH but poor labeling of human cells.
    • Monobromobimane provided reproducible staining of human cells with manageable background fluorescence (approx. 20%).
    • Mercury orange and other tested stains exhibited less consistency or unacceptably high background fluorescence.

    Conclusions:

    • Monobromobimane is the preferred stain for quantifying cellular GSH in human cells using flow cytometry.
    • Careful control of stain concentration and incubation time is essential for monobromobimane.
    • Routine inclusion of GSH-depleted control samples is recommended for accurate background assessment in flow cytometric GSH assays.