Quantitative Determination of Intracellular GSH

Cell lysates were collected and immediately subjected to protein thiol and GSH measurement as reported (41 (link), 42 (link), 43 (link)). Fluorescent intensities were detected at 400Ex/465Em by CLARIOstar microplate reader (BMG LABTECH). Quantitative determinations of GSH and GSSG levels were performed using the enzymatic recycling method. Briefly, protein in the cell extracts was precipitated by sulfosalicylic acid, and the supernatant was then divided into two parts. For reduced GSH, the supernatant was incubated with thiol fluorescent probe IV (Sigma; #595504), and fluorescence intensities were measured. For total GSH (GSH + GSSG), the supernatant was neutralized by triethanolamine and incubated with the reduction system, containing NADPH (Sigma; #10107824001) and GSH reductase (Sigma; #10105678001), at 37 °C for 20 min. GSSG was calculated based on the results from reduced GSH and total GSH; the ratio of GSH/GSSG = [GSH]/(([Total GSH] − [GSH])/2).