Glutathione gsh gssg total fluorometric assay kit

Following the indicated treatments, HCT116 and C26 cells were lyzed, and the ATP content in the supernatant was measured with an ATP Assay Kit (Beyotime Biotechnology, Shanghai, China). The values were normalized to the protein concentration. The GSH/GSSG ratio was determined using the Glutathione (GSH/GSSG/Total) Fluorometric Assay Kit (BioVision, CA, USA).

An NADP/NADPH-Glo assay (Promega) was used to measure NADPH/NADP⁺ levels, according to manufacturer's instructions. Briefly, cells were seeded in 6-well plates (1 × 10⁵/well for MAD11 and 2 × 10⁵/well for HEC1A) and treated for 2.5 h with BH10 (20 μM) or equivalent volumes of vehicle control (DMSO). Media was removed from wells prior to adding equal volumes (60 μL) of PBS then 1% dodecyltrimethylammonium bromide in 0.2 N NaOH. The remainder of the assay followed the protocol supplied with the kit. Luminescent signal was detected using an EnSight Multimode plate reader (PerkinElmer). GSH:GSSG ratios were measured using the Glutathione (GSH/GSSG/Total) Fluorometric Assay Kit (Biovision, K264). Cells were seeded in 10 cm dishes (1 × 10⁶ for MAD11 and 2 × 10⁶ for HEC1A) in 10 mL media. The following day, cells were treated with BH10 (20 μM) or equivalent volumes of vehicle (DMSO) for 2.5 h. Cells were rinsed with ice-cold PBS and then scraped from the dish in 800 μL ice-cold PBS. Cells were spun at 5000 rpm for 3 min at 4 °C, cell pellets resuspended in 100 μL glutathione assay buffer, and ice-cold perchloric acid (20 μL) was added to each sample (60 μL). GSH and GSSG levels were then measured according to manufacturer's instructions.