Figure 6: Intracellular ROS levels in NB4 cells treated with esculetin and t-BHP. Cells (5 × 105/ml) were treated with 100 µM esculetin or 100 or 500 µM t-BHP or the combination of both, for the indicated times, and then analyzed by flow cytometry using a specific probe. (a) To measure intracellular superoxide levels, 2 μM DHE was added during the last 15 min of the esculetin treatment. (b) To quantify intracellular peroxides, 10 μM DCFDA was added for 30 minutes at 37°C after the treatments. One representative experiment is shown out of three. Vertical axes: number of events (linear scale); horizontal axes: fluorescence intensity (log scale). F.I. represents mean fluorescence intensity.