2.9.3. GSH/GSSG Ratio

The GSH/GSSG-Glo™ assay (Promega Co., Madison, WI, USA) was used to measure the ratio. RAW264.7 cells were cultured in a 96-well plate containing a medium. After 3 h, the old medium was removed, and either DMEM (control group), 2 μg·mL⁻¹ LPS (positive control group), or broccoli extract with different concentrations of SFN were added, and the cells were cultured for 20 h. Total glutathione reagent (50 μL·well⁻¹) or oxidized glutathione reagent (50 μL·well⁻¹) was added to each well and shaken for 5 min. In addition, the glutathione standard was diluted into 8 different concentrations by a 2-fold serial dilution method, and the total glutathione reagent (50 μL·well⁻¹) was added. Luciferin generation reagent (50 μL·well⁻¹) was added to each treatment group and the standard group and mixed well and incubated at room temperature for 30 min. Next, the luciferin detection reagent (100 μL·well⁻¹) was added and allowed to stand for 15 min, and the luminescence was measured (integration time = 0.3 s). The GSH/GSSG ratio is calculated as follows: ratio GSH/GSSG treated = (μM total glutathione treated – (μM GSSG treated × 2))/μM GSSG treated.