Gsh glo glutathione assay kit

Cells were seeded in 96-well plates at 2 × 10⁴ cells per well and incubated for 12 h. The medium was replaced by treatment medium. After 24 h of treatment, GSH levels in cells were detected using a GSH-Glo™ Glutathione Assay kit (Promega, St Louis, MO, USA) according to the manufacturer’s instructions. GSH levels were normalized by total protein levels and were expressed as percentage of the untreated control. The total protein levels were measured with a BCA assay kit.

Primary microglia (5 × 10⁴ per well) were seeded overnight in clear-bottom black 96-well plates to allow cells to adhere, incubated in serum-free medium, and treated with LPA (1 or 5 µM) for the indicated time periods. To measure intracellular glutathione content, cells were incubated with the GSH-Glo™ reagent (GSH-Glo™ Glutathione Assay Kit; Promega Corporation, Madison, WI, USA) for 30 min. After addition of the luciferin detection reagent, luminescence was measured to quantify the glutathione content according to the manufacturer’s protocol.

GSH (10 mM) was incubated with 3 µg of the RipAY or YER163c protein for 60 min at 30°C in 100 µl of reaction mixture containing 50 mM Tris-HCl (pH 8.0). The reaction mixture was then incubated at 95°C for 5 min to inactivate the enzyme. Yeast and plant extracts and E. coli lysates were added to the reaction mixture when necessary for each assay. The GSH level of the reaction mixture was measured with the GSH-Glo glutathione assay kit (Promega). Yeast extracts were prepared as described below. Plant extracts were prepared as follows. Briefly, leaf disks taken from Arabidopsis Col-0 plants were frozen in an Eppendorf tube in liquid nitrogen and then ground by shaking with tungsten beads using an SH-48 sample smasher (Kurabou, Osaka, Japan). Then 50 mM Tris-HCl (pH 8.0) buffer was added to the samples, and the mixture was thoroughly vortexed. Cell debris was removed by centrifugation, and the resulting supernatant was collected in new tubes and used for the assay as a plant extract. The purified recombinant proteins of yeast TRX2 thioredoxin (Oriental Yeast, Tokyo, Japan) and E. coli thioredoxin (Sigma, St. Louis, MO) were added to the reaction mixture at appropriate concentrations.

Cellular GSH contents were measured using a GSH-Glo™ Glutathione Assay kit (Promega, WI, USA) according to the protocol provided by the manufacturer.