Poly l lysine (pll)

Cortical neurons were prepared from 16-day-old Sprague Dawley rat embryos (Charles River Laboratories Japan, Inc., Yokohama, Japan). Cortices were dissociated using the Papain Dissociation System (Worthington Biochemical Corporation, Lakewood, NJ, USA) according to the manufacturer’s instructions, and the cells were seeded onto 96-well plates pre-coated with poly-L-lysine (AGC Techno Glass Co., Ltd., Shizuoka, Japan) at a cell density of 5.0 × 10⁴ cells/well. The cells were placed in a 5% CO₂ incubator at 37°C and cultured for 20 days in the Neurobasal Plus Medium supplemented with 2% B27 Plus Supplement and 40 μg/mL gentamicin (all from Thermo Fisher Scientific Inc., Waltham, MA, USA). The culture medium was exchanged every 3–4 days and removed just before the application of NMDA. The cells were simultaneously incubated for 2 h with the test compounds and NMDA, and the cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays in accordance with the manufacturer’s instructions (CellTiter 96 AQ_ueous One Solution Cell Proliferation Assay, Promega Inc., Madison, WI, USA).

HTM cells were seeded in 96-well plates pre-coated with poly-L-lysine (AGC Techno Glass Co., Ltd., Shizuoka, Japan) at a density of 1.0 × 10⁴ cells/well. The cells were placed in a 5% CO₂ incubator at 37°C and cultured in supplemented TMCM. The HTM cells reached confluence at 24 h after seeding, following which, the supplemented TMCM was removed and the cells were washed once with non-supplemented TMCM. The cells were incubated for 24 h with OMD at 10 nM, 100 nM, and 1 μM in non-supplemented TMCM containing 0.1% DMSO, while non-supplemented TMCM containing 0.1% DMSO was used as the vehicle, and the cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays in accordance with the manufacturer’s instructions (CellTiter 96 AQ_ueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA).