4.8. Measurement of Intracellular ROS Accumulation

Intracellular H₂O₂ levels were determined by measuring the fluorescence of DCFH-DA. For the purpose of these experiments, the cells were plated in six-well culture plates with coverslips. The cells were treated with IL-1β with/without pretreatment with RTA 408 for the indicated time intervals (5 and 10 min). The cells were washed with warm PBS and incubated in PBS containing 10 μM DCFH-DA at 37 °C for 30 min. Subsequently, the PBS containing DCFH-DA was removed and replaced with fresh medium. The cells were washed twice with PBS and then observed by using a fluorescence microscope (Zeiss, Axiovert 200M). In addition, CellROX Deep Red Reagent was added to the cells at a final concentration of 5 μM and then incubated for 30 min at 37 °C. Subsequently, the medium was removed, and the cells were washed three times with PBS. The resulting fluorescence was measured using a fluorescence microscope.