2.9. Cell Culture and MTT Assay

SH-SY5Y neuroblastoma cell line was obtained from Korean cell line bank (Seoul, South Korea) and grown in EMEM (ATCC, Manassas, Virginia, United States)/F12 medium (Gibco, Carlsbad, CA, USA) (1:1) supplemented with 10% FBS (GE Healthcare, Chicago, IL, USA) and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA) and cultured in 5% CO₂ at 37 °C. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA) assay [30]. SH-SY5Y cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well and cultured for 24 h. To assess the cytotoxicity, SH-SY5Y cells were treated with CC or FCC (20, 50, and 100 μg/mL) for 24 h. For cell viability, SH-SY5Y cells were pretreated with CC or FCC for 2 h and then co-incubated with 300 µM H₂O₂ for another 24 h. The 300 µM H₂O₂ was freshly prepared from a 30% (mass/mass) stock solution. The concentration and molar mass of the stock solution is 1.150 g/mL and 10.014 M. The stock solution was first diluted to 40 mM and then diluted to 300 µM in the medium. Morphologic images of SH-SY5Y cells were taken by an inverted microscope (Olympus, Tokyo, Japan) at fixed 400× magnification.