Arpe 19 cells crl 2302

The RPE cell line, ARPE-19 cells (CRL-2302), was purchased from the American Type Culture Collection (ATCC^®: Manassas, VA, USA) and used in this study. ARPE-19 cells culture used 10% fetal bovine serum (FBS: Biosera, Kansas City, MO, USA) contained Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 (FUJIFILM-Wako), containing 100 μg/mL streptomycin (Meiji, Tokyo, Japan) and 100 U/mL penicillin (Meiji) as the antibiotic. ARPE-19 cells were passaged every 4 days by trypsinization, and these were cultured in an incubator humidified atmosphere containing 5% CO₂ at 37 °C. In all experiments, ARPE-19 cells with passage numbers 5–9 were used. The cells were seeded at a density of 1.5 × 10⁵ cell/mL in an 8-well chamber or 96-well plate depending on the experiment. All experiments were performed after the cells were seeded and cultured for 4 days to ensure that the cells reached confluence.

ARPE-19 cells (CRL-2302) were purchased from the American Type Culture Collection (Manassas, VA, USA) and routinely cultured in Dulbecco’s Modified Eagle Medium/F-12 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (WELGENE Inc., Gyeongsan, Republic of Korea) as described previously [27 (link)]. To investigate beneficial effects of phloroglucinol on oxidative damage, cells were cultured in media containing desired concentrations of phloroglucinol and H₂O₂ (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 24 h or pretreated with phloroglucinol, N-acetyl-L-cysteine (NAC), Mito-TEMPO, and/or 3-methyladenine (3-MA, Sigma-Aldrich Co., St. Louis, MO, USA) for 1 h prior to treatment with H₂O₂ for 24 h. To investigate the blocking effect of phloroglucinol on the generation of ROS induced by H₂O₂, cells were pretreated with phloroglucinol, NAC, and Mito-TEMPO for 1 h and then treated with H₂O₂ for 1 h. To acquire fluorescence images of ROS generation, γH2AX expression, and autophagic vacuoles, cells cultured on coverslips were stimulated with H₂O₂ in the presence or absence of phloroglucinol, NAC, and/or Mito-TEMPO. After treatment, cells were washed with phosphate-buffered saline and subjected to fluorescence staining.