Measurement of ROS levels
After treatment with different concentrations of icariin for 24 h, the cells were harvested and incubated with DCFH-DA for 20 min at 37ºC. The ROS levels of treated cells were measured via flow cytometry (FCM). For inhibition experiments, cells were treated with an antioxidant (NAC, 2 mM) for 1 h prior to icariin exposure.
Mitochondrial membrane potential (ΔΨm) assay
Rh123 was exploited to measure the changes in the ΔΨm of cells via FCM. Cells were treated with different concentrations of icariin and then incubated with Rh123 (5 μg/mL) at 37ºC for 30 min. Finally, the ΔΨm of the treated cells was measured by FCM.