Glutathione Assay for Oxidative Stress

The oxidative stress assessment was evaluated using the GSH-Glo Glutathione Assay kit (Promega, WI, USA). For the GSH-Glo Assay analysis, AFSC were seeded at a density of 3000 cells in 300 µL of Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin, streptomycin/neomycin) in 96-well plates. After 24 h of seeding, cells are treated with the studied samples and then incubated for 72 h. To the obtained solution, 100 µL 1 × GSH-Glo Reagent was added, followed by incubation at 37 °C for 30 min and then 100 µL Luciferin Detection Reagent was added and incubated at 37 °C for an additional time of 15 min. The medium from the wells was well homogenized and then the plate was read on the luminometer (Microplate Luminometer Centro LB 960, Berthold, Germany) [37 (link),38 (link)].