Fluorescent probes

Fluorescent probes (Beyotime Technology, Shanghai, China) were diluted in serum‐free medium to a final concentration of 10 μmol/L. PC12 cells were then cultured in 96‐well plates and divided into two groups (GF+DMSO and GF+5‐PAHSA), as previously above. After 24 h of intervention, the culture media were removed and the Fluorescent probes were added. After 20 min of incubation at 37°C and 5% CO₂, the cells were washed three times in serum‐free medium. Finally, fluorescence intensity was detected with a fluorescence microplate reader (Bio Tek Synergy H4, Winooski, USA).

Human retinal pigment epithelial cells (ARPE-19) were maintained in Dulbecco’s modified Eagle’s medium/Ham’s F12 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 IU/mL penicillin (Gibco), and 100 μg/mL streptomycin (Gibco) at 37 °C in an atmosphere of 5% CO₂. After incubation, cells were exposed to 200 μM H₂O₂ for 24 h, and fluorescent probes (Beyotime Biotechnology, China) were used to measure intracellular ROS levels (DCFH-DA, 10 mM) according to the manufacturer’s instructions. Before application, CBLs and P-CBLs of a certain volume were diluted with complete culture medium. The final concentration of BBH was 0.0040 μM and 0.0020 μM for CHR in CBLs groups, while 0.0044 μM of BBH, 0.0018 μM for CHR for P-CBLs, respectively. The final drug concentration of CBs was the same as CBLs group. Treated cells were loaded with fluorescent probes in a serum-free medium for 20 min at 37 °C. Then, photographs were taken using a long-term real-time dynamic live cell imaging analyzer.