A cytochemical stain for glutathione in rat hepatocytes cultured on plastic

Thiol groups of glutathione react with the organomercurial azo dye mercury orange at a faster rate than with -SH groups of proteins. This property makes possible visualization of glutathione in cells without appreciable interference from other -SH groups. To render this method useful for cytochemical localization of glutathione in plastic cultured cells, it was necessary to adapt this reaction to the specific characteristics of the biological samples to be assayed. First, the choice of a solvent that would allow a convenient solubility of the dye and at the same time be compatible with the plastic culture plate was crucial. Second, to avoid diffusion of glutathione out of the cell the procedure for staining cells was also important. Satisfactory results were obtained after 30-40 sec reaction with 50 microM mercury orange in acetone/water 9:1, v/v, at room temperature. Glutathione-mercury orange complexes exhibited orange fluorescence on excitation with blue light. No diffusion of glutathione out of the cells was observed, and the hepatocytes stained with the dye showed orange fluorescence which paralleled their glutathione content.