Measurement of Cellular Glutathione

Cells were harvested by washing with and scraping into ice-cold phosphate-buffered saline (PBS). The cellular glutathione concentrations were measured enzymatically, as described previously by Tietze (1969) using the GSH/GSSG-412 kit. Briefly, samples were prepared in 5% metaphosphoric acid to remove proteins. Ellman’s reagent (5,5'-dithiobis-2-nitrobenzoic acid or DTNB), which reacts with reduced glutathione, was used to form a colored product spectrophotometrically detectable at 412 nm. Oxidized glutathione was recycled into reduced glutathione by glutathione reductase and β-nicotinamide adenine dinucleotide phosphate (NADPH). The rate of this reaction found by measuring the change in the absorbance was a linear function of the glutathione concentration in the reaction mixture. The parameters of a six-point calibration curve were used for glutathione concentration determination.