Neuronal Differentiation of SH-SY5Y Cells

SH-SY5Y cells are often employed as the in vitro models for anxiety [40 (link)] and pain [33 (link),41 (link)]. The SH-SY5Y cell lines (ATCC, Manassas, VA, USA) were maintained in DMEM/Ham’s F12 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 2.5 mM L-glutamine, 10% fetal bovine serum, and antibiotics—100 U/mL of penicillin and 100 μg/mL of streptomycin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)—in a humidified atmosphere of 5% CO₂ at 37 °C. For the neuronal induction of SH-SY5Y cell lines, the culture medium was replaced with a differentiation medium containing 10 μM of all-trans retinoic acid (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) in the presence of Rice-koji extracts (10 or 500 µg in 1 mL PBS), EGT (10 or 100 µg in 1 mL PBS), or PBS alone. The differentiation medium was replaced every 5 days over 4 weeks of incubation [42 (link)].