2.4. Measurement of ROS

The Reactive Oxygen Species Assay Kit using fluorescent probe DCFH-DA was applied to detect the level of intracellular reactive oxygen species. Based on the instructions, after 24 h exposure, we washed the HCEC in the 6-well plate with PBS twice to remove the exposure fluid, then treated cells with 1 mL fluorescent probe DCFH-DA at 10 μM each well and incubated for 20 min at 37 °C. Next, we rewashed them with serum-free DMEM/F-12 medium and digested cells with trypsin to measure ROS production by flow cytometry (CyFlow® Cube 6, Patec, Nuremberg, Germany). Each exposure group was tested for fluorescence with a density of 1 × 10⁴ cells.