High Throughput Method for Assessment of Cellular Reduced Glutathione in Mammalian Cells

Reduced glutathione (GSH) is an intracellular molecule essential for many aspects of cell physiology and defense. Determination of GSH has been used to identify potential anti-cancer drugs and for the assessment of drug toxicity via generation of oxidative stress. The described protocol was designed to modify existing protocols for the fluorescent detection of intracellular GSH in a high throughput 96-well microplate format. Dibromobimane was used to label intracellular GSH, and an additional dye, Hoechst 33342 was used to measure cell density for data normalization. Cell density curves were performed using HEK 293T cells to determine the optimal starting cell density, (< 8.0 × 10(4) cells/well) for fluorescent analysis. Fluorescent dyes were also analyzed for compatibility and spectral overlap. The method was further validated by exposing HEK 293T cells to GSH modulating agents; tert-butylhydroquinone a potent inducer of GSH, and L-buthionine-(SR)-sulfoximine a potent inhibitor of GSH. This study provides a fast, simple method for the high throughput screening of GSH in a widely available 96-well format. It also addresses the pitfalls associated with fluorescent compounds in cell culture and proper data normalization.