John Shultz, Mary Sobol, Nancy Murphy, Jolanta Vidugiris
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711
A wide variety of compounds can induce intracellular formation of reactive oxygen species (ROS) and the resulting cellular damage can eventually lead to apoptosis or necrosis. Measuring the ratio of reduced to oxidized glutathione (GSH/GSSG ratio) is a way to determine how such compounds alter the redox potential in the cell. A new method is presented for rapid determination of the GSH/GSSG ratio in mammalian cells in multiwell tissue culture plates. The method is based on the formation of luciferin from a glutathione S-transferase catalyzed reaction using GSH and a luciferin precursor. The method utilizes a cell lysate generated directly in the wells of the cell culture plate and does not require protein removal steps. In the reaction to measure total glutathione, DTT is added to convert all the glutathione in the reaction to the reduced form. In the reaction to measure oxidized glutathione (GSSG), free sulfhydral groups are inactivated with NEM leaving the GSSG intact. The inactivation step is followed by a reducing step that converts the GSSG to GSH for quantification in the luminescent reaction. Since the reaction of free sulfhydral groups takes place immediately upon cell lysis, additional formation of GSSG from air oxidation of GSH is prevented. The assay takes less than two hours from the time a plate of cells is ready for measurement and can be performed using less than 1000 cells per well. The performance of the method is demonstrated by following the effects of compounds such as menadione, ethacrynic acid and chlorodinitrobenzene on the levels of GSH and GSSG in cells. This is a fast, easy, and sensitive assay for identification of compounds that alter the GSH/GSSG ratio.