Glutathione assay

The relative glutathione (GSH) concentration in cell or tissue lysates was assessed using the glutathione assay kit (CS0260; Sigma) according to the manufacturer’s instructions. Cells were seeded onto 10 cm plate (5 × 10⁶ cells per plate) and treated with DMSO, or erastin for 24 h. Cells were washed with ice-cold PBS and lysed in 1% lysis buffer (25 mM Tris at pH 7.5, 300 mM NaCl, 1 mM EDTA, 0.5% NP-40) supplemented with a phosphatase inhibitor mix (Pierce) and a complete protease inhibitor cocktail (Roche). After sonication (Thermo Model 120, amplitude 15%, process time 10 s, push-on time 5 s, and push-off time 1 s), cell lysates were centrifuged at 13,200 rpm at 4 °C for 10 min, and cleared lysate was used to determine the amount of GSH in the sample. Enzymes were removed by using deproteinizing sample kit (ab204708), which can interfere with the analysis. Add 50 µL of GSH assay mixture into each sample well and incubate it at room temperature for 60 min protected from light. The measurement of GSH used a kinetic assay in which catalytic amounts (nmoles) of GSH caused a continuous reduction of 5,5′-dithiobis (2-nitrobenzoic acid) to 5-thio-2-nitrobenzoic acid. The reaction rate was proportional to the concentration of glutathione up to 2 mM. The yellow product (5-thio-2-nitrobenzoic acid) was measured spectrophotometrically at 412 nm.