Sm1 components

Primary cultures of rat hippocampal neurons were prepared from the brains of day 18 embryonic rats. Briefly, the hippocampus was dissected in free HBSS and incubated with a 0.125% trypsin solution for 15 min at 37 °C. The resulting cell suspensions were diluted in neurobasal medium (#21103–049, Gibco), supplemented with SM1 components (#05711, Stemcell), and plated onto 100 μg/mL poly-D-lysine (#P0899, Sigma-Aldrich) and 2 μg/mL laminin (#11–243–217-001, Roche)-coated plates or coverslips.

The hippocampal neuronal cell line HT22 was maintained in Dulbecco’s modified Eagle’s medium (#11995-065, Gibco, Waltham, MA, USA) supplemented with 10% (v/v) FBS, 20 mM HEPES (pH 7.2), and 1% penicillin-streptomycin (#15140122, Gibco, Waltham, MA, USA) in a CO₂ incubator at 37 °C. Primary hippocampal neurons were cultured from embryonic day 18 Sprague–Dawley fetal rats, as previously described [52 (link)]. Briefly, the hippocampal tissue was dissected in Hanks’ Balanced Salt solution (#14185052, Gibco, Waltham, MA, USA) and dissociated with 0.125% trypsin (#15090046, Gibco, Waltham, MA, USA) for 15 min at 37 °C. Cells were seeded on a pre-coated coverslip or plate with 100 μg/mL poly-D-lysine (#P6407, Sigma-Aldrich, St. Louis, MO, USA) and 10 μg/mL laminin (#354232, Corning, Lowell, MA, USA). The hippocampal neurons were incubated in a Neurobasal medium (#21103-049, Gibco, Waltham, MA, USA), 0.5 mM glutamine, and 25 μM glutamate and supplemented with SM1 components (#05711, Stemcell, Vancouver, BC, Canada) for 18–21 days in vitro.