Differentiation of SH-SY5Y Neuroblastoma Cells

The SH‐SY5Y human neuroblastoma cell line (ATCC^® CRL‐2266^TM) was purchased from the American Type Culture Collection. Cell culture and differentiation were performed as described previously (Tagai et al., 2020 (link)). In brief, SH‐SY5Y cells were cultured in Dulbecco's modified eagle medium (D‐MEM)/Ham's F‐12 (FUJIFILM Wako Pure Chemical Corporation, Cat#048‐29785) containing 10% fetal bovine serum (FBS), 100‐units/ml penicillin, and 100‐μg/ml streptomycin in a 37°C incubator in an atmosphere of 5% CO₂ and 100% relative humidity. In the cell differentiation, SH‐SY5Y cells were inoculated at an initial density of 1.0 × 10⁵ cells/dish in a collagen I‐coated φ 3.5 cm dishes. All‐trans‐RA (FUJIFILM Wako) was added 2 days after plating at a final concentration of 10 μM dissolved in high‐glucose D‐MEM (FUJIFILM Wako, Cat#043‐30085), which was supplemented with 15% FBS. After 5 days of culturing in the presence of RA, cells were washed with high‐glucose D‐MEM and incubated for 2 days with 100 ng/ml human recombinant BDNF (FUJIFILM Wako) in high‐glucose D‐MEM without L‐glutamine and phenol red (FUJIFILM Wako, Cat#040‐30095) containing 4 mM sodium pyruvate and 1 mM L‐glutamine. We prepared ndSH‐SY5Y cells using a modified differentiation protocol according to previously described methods (Agholme et al., 2010 (link); Encinas et al., 2000 (link); Krishtal et al., 2017 (link)).