Dcfda h2dcfda cellular ros assay

To evaluate the antioxidant capacity of the SF extracts in cell culture, the DCFDA/H2DCFDA–Cellular ROS Assay (Abcam, Cambridge, UK) was employed. SH-SY5Y cells were plated at the density of 25,000 cells per well in black, clear-bottomed 96-well plates. The next day, cells were incubated with 20 μM 2',7'-dichlorofluorescin diacetate (DCFDA) for 45 min at 37°C in the dark and then treated with the SF extracts at various concentrations in the presence of 250 μM tert-butyl hydroperoxide (TBHP) for 4 hours. DCFDA is a nonfluorescent probe that is oxidized in the highly fluorescent 2',7'-dichlorofluorescein (DCF) in the presence of ROS [39 (link)]. DCF fluorescence was measured in a Synergy H1 microplate reader (BioTek, Vermont, USA) at Ex/Em = 485/535 nm. Trolox (500 μΜ) was employed as standard antioxidant.

BMDMs were seeded in 96-well plate (50,000 cells/well) for 24 h. Cells were primed with Pam3CSK4 for 24 h then incubated with either fingolimod phosphate (2.5 μM), febuxostat (200 μM) or NAC (10 μM) for 3 h. Finally, cells were stimulated with MSU crystals (250 μg/ml) for 3 h. ROS generation was measured using DCFDA/H2DCFDA cellular ROS assay (Abcam) according to manufacturer’s instructions.