Figure 4: Intracellular ROS levels in NB4 cells treated with esculetin and H2O2. Cells (5 × 105/ml) were treated with 100 M esculetin, 1 mM H2O2, or the combination of both, for different 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 analyze intracellular peroxides, 10 μM DCFDA was added for 30 minutes at 37°C after the treatments. One representative experiment is shown out of five. Vertical axes: number of events (linear scale); horizontal axes: fluorescence intensity (log scale). F.I. represents mean fluorescence intensity.