Measurement of intracellular reactive oxygen species (ROS)

To obtain further evidence for the protective effect of CGA against H₂O₂ induced oxidative stress, alterations of intracellular ROS levels were determined. The production of intracellular ROS was measured using 2′,7′-dichlorofluorescin diacetate (DCFH-DA; Invitrogen, Carlsbad, CA, USA) by flow cytometry. Briefly, hLECs were incubated either with 100 µM H₂O₂ alone or treated with different concentrations (10, 30 and 50 µM) of CGA for 2 h prior to treatment with 100 µM H₂O₂ for 24 h. After harvest of hLECs, the cells were incubated with DCFH-DA solution (10 µM) in the dark at 37°C for 30 min, washed with PBS (pH 7.4), and analyzed within 30 min using a flow cytometer (Accuri C6; Accuri Cytometers Inc., Ann Arbor, MI, USA). The specific fluorescence signals that correspond to DCFH-DA were collected with a 525 nm band pass filter. For each determination, 2.0×10⁴ cells were counted.