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Related Experiment Videos

Flow cytometry techniques for studying cellular thiols.

R E Durand, P L Olive

    Radiation Research
    |September 1, 1983
    PubMed
    Summary
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    Quantifying cellular thiols, like glutathione, helps assess damage risk from radiation and carcinogens. This study developed a rapid assay using fluorescent probes for viable cells, improving understanding of thiol-mediated protection.

    Area of Science:

    • Biochemistry
    • Cell Biology
    • Radiation Biology

    Background:

    • Cellular thiols, particularly glutathione, are crucial nucleophiles protecting against damage from ionizing radiation and carcinogens.
    • Assessing cellular thiol levels can predict susceptibility to such damage.
    • Existing fluorescent thiol probes often lack specificity for protein vs. nonprotein thiols and require cell fixation.

    Purpose of the Study:

    • To develop and apply a rapid assay for quantifying thiol content in viable cells.
    • To investigate the spatial distribution of thiols within multicellular spheroids.
    • To correlate cellular thiol levels with radiation response and the efficacy of thiol-modulating agents.

    Main Methods:

    • Utilized fluorescent thiol-reactive probes that penetrate and bind to thiols in viable cells.

    Related Experiment Videos

  • Employed flow cytometry and fluorescence-activated cell sorting (FACS) for thiol quantification.
  • Assessed radiation response of sorted cells after treatment with exogenous thiols (glutathione, WR-2721) or thiol-depleting agents (BSO, DEM, DMF).

    • Main Results:

    • Identified fluorescent probes capable of rapid, low-cytotoxicity binding in viable cells, quantifiable by flow cytometry.
    • Mapped thiol content variation across different depths of V79 spheroids using FACS.
    • Demonstrated that cellular thiol levels directly influence radiation sensitivity and the protective effects of exogenous thiols.

    Conclusions:

    • The developed assay provides a rapid method to quantify cellular thiols in viable cells, aiding in risk assessment.
    • Spatial analysis within spheroids reveals heterogeneity in thiol content and radiation response.
    • Understanding thiol-cell interactions is key to elucidating mechanisms of radioprotection and carcinogen detoxification.