ROS generation was detected using the ROS-Glo™ assay (Promega, Southampton, UK) according to the manufacturer's instructions (39 (link)). Briefly, cells were stimulated for 2 h with Pam3, MSU, or soluble uric acid in the presence of 25 μM ROS-Glo H2O2 substrate. Stimulations were completed in serum-free Optimem cell culture media (Gibco, ThermoFisher). After 2 h incubation at 37°C and 5% CO2, ROS levels were determined following a 20 min incubation with the ROS-Glo™ detection reagent. Luminescence was measured by spectrophotometry (Biotek Synergy HT).
N-acetyl-L-cysteine (NAC) was used in some experiments to inhibit ROS in primary human monocytes. For these experiments, cells were preincubated for 1 h with NAC and then stimulated for 6 h with Pam3 ± MSU crystals. NAC concentration was maintained during the 6 h stimulation. ROS levels were determined by ROS-Glo™ and IL-1β secretion by ELISA.
N-acetyl-L-cysteine (NAC) was used in some experiments to inhibit ROS in primary human monocytes. For these experiments, cells were preincubated for 1 h with NAC and then stimulated for 6 h with Pam3 ± MSU crystals. NAC concentration was maintained during the 6 h stimulation. ROS levels were determined by ROS-Glo™ and IL-1β secretion by ELISA.
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