Sh sy5y cells ecacc

SH-SY5Y cells (ECACC, Sigma Aldrich, St. Louis, MO, USA) were cultured in a 1 : 1 mixture of Ham's F12 and Dulbeco's modified Eagle's medium (DMEM) (Gibco Life Technologies, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone Labs., Logan, UT) and 100 U/mL of penicillin/streptomycin (Gibco Life Technologies, Grand Island, NY, USA) in a humidified atmosphere of 5% CO₂ at 37°C. The medium was changed twice a week, and cells were split at about 80% confluence. For neuronal differentiation, 5 × 10⁴ cells/well was seeded in collagen-coated plates (100 μg/mL, Corning, USA). After 24 hours (day 1), the medium was replaced by medium in which FBS concentration was reduced to 2% (differentiation medium) and supplemented with 10 μM all-trans retinoic acid (RA, Sigma Aldrich, Saint Louis, MO). Cells were incubated for 5 days, with the medium replaced every day, except on the second day. At day 5 of differentiation, the medium was removed, and cultures were stimulated with serum-free medium supplemented with human brain derived neurotrophic factor (BDNF 50 ng/mL—R&D Systems, MN, USA). At day 7 of differentiation, neurons were used in the experiments.

The human neuroblastoma SH-SY5Y cells (ECACC, #94030304) were purchased from Sigma-Aldrich (Milan, Italy). SH-SY5Y cells were cultured in polystyrene-coated flasks in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 mg/L streptomycin (Sigma-Aldrich). Cultures were maintained at 37 °C in 5% CO₂ and medium was changed twice per week. When flasks were at about 80% confluency, the culture was passaged at a ratio of 1:10. Only cultures between passages 3 and 13 were used.
In separate experiment, differentiation to a more dopaminergic phenotype was carried out using the previously reported protocol [90 (link),91 (link)]. In brief, when cells were at 75% confluency, medium was changed for new fresh 1% FBS medium and 10 µM retinoic acid. After three days, medium was changed for fresh 1% FBS medium and 80 nM phorbol ester, and cells were incubated for an additional three days.