Measurement of intracellular ROS levels

The fluorescent agent, 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA; Shanghai Yeasen Biotech Co., Ltd.) was used to investigate ROS production. The cells were seeded in 6-well plates at 37°C in a humidified atmosphere with 5% CO₂, at a density of 5×10⁵ cells/well. After incubation for 24 h, the cells were treated with FYGL at a concentration range of 0–500 µg/ml for 24 h. Following which, the cells were washed with PBS and added to 1 ml serum-free medium with 10 µl DCFH-DA per well. After incubation for 30 min at 37°C, the level of ROS in the cells were identified using DCFH, and analyzed using flow cytometry (Beckman Coulter, Inc.), and the data were analyzed using FlowJo software (version 10.4; BD Biosciences).