Flow cytometry techniques for studying cellular thiols.

Cellular thiols, and especially glutathione, act as scavenger nucleophiles and can protect against toxicity, mutagenicity, or transformation by ionizing radiation and many carcinogens. Development of a rapid assay to quantitate the cellular content of thiols could thus be useful in assessing or predicting cellular risk to damage. Several fluorescent thiol-reactive drugs, usually maleimide or bromobimane derivatives, have been described for use in histopathology. Most of these agents do not distinguish between protein and nonprotein thiols, and virtually all of these fluorescent stains have normally been used after fixation of the cells or tissues. We have found that some of the probes will, however, rapidly penetrate and bind within viable cells with little associated cytotoxicity; the amount bound can be easily quantified using flow cytometry. We have used several of these agents, in conjunction with fluorescence-activated cell sorting in V79 spheroids, to examine the thiol content of cells as a function of their depth or position in the spheroid. Additionally, the radiation response of cells from different depths has been assessed following addition of exogenous thiols including glutathione and WR-2721, or after treatment with thiol-depleting agents, including DL-buthionine-S,R-sulfoximine (BSO), diethylmaleate (DEM), and dimethylfumarate (DMF). Our studies indicate that examination of the thiol content and radiation response of the sorted cells provides an improved understanding of the modes of action of these compounds.