Sumitomo nerve cell culture system

Primary cultures of hippocampal neurons were prepared from neonatal (P0) C57BL/6JJ mice using the SUMITOMO Nerve-Cell Culture System (Sumitomo Bakelite, Tokyo, Japan). Hippocampal neurons were then cultured in Neurobasal Medium (Thermo Fisher Scientific) containing 2% B-27 Supplement (Thermo Fisher Scientific) at 37 °C and 5% CO₂, replacing half the volume of the medium every 5 days.
PC12 rat pheochromocytoma cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 10% horse serum (HS), 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C and 5% CO₂ on collagen-coated dishes. For neurite outgrowth, PC12 cells were cultured in DMEM containing 50 ng/mL NGF, 0.5% FBS and 0.1% HS, 100 units/mL penicillin, and 100 μg/mL streptomycin.

E16.5 pregnant mice of 1.5 Mb patDp+ were used to prepare primary neuronal culture. Embryos were surgically removed and hippocampi were dissected in ice-cold Hank’s balanced salt solution. Then, primary hippocampal neurons were prepared by using SUMITOMO Nerve-Cell Culture System (Sumitomo Bakelite Co., Ltd). Prepared neurons were maintained in the neuronal plating medium containing MEM, 5% fetal bovine serum, 2 mM L-Glutamine, 0.6% D-glucose, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C, 5% CO₂ for 6 h. The medium was changed to neuronal maintenance medium (Neurobasal/NeuroBrew-21 supplement, 0.5 mM L-glutamine) until analysis. Neurons were plated on a poly-L-lysine-coated six-well plate at the density of 5 × 10⁵ cells/well. On the day in vitro 7, neurons were washed with PBS and lysed in TRIZOL Reagent (Thermo Fisher Scientific) to extract total RNA.

Primary cultures of hippocampal neurons were prepared as previously described with a few modifications [18 , 19 (link)]. In brief, hippocampi were dissected from mice aged from E17 to P4. Hippocampal neurons were dissociated by using SUMITOMO Nerve-Cell Culture System (Sumitomo Bakelite) and plated on coverslips or glass-based dishes coated with poly-D-lysine at a density of 1.0–2.0 x 10⁵ cells/cm² in MEM supplemented with 10% horse serum (Gibco), 0.6% D-glucose, 1 mM sodium pyruvate and 1% penicillin-streptomycin. Three hours after plating, media was replaced by Neurobasal medium (Gibco) supplemented with B-27 supplement (Gibco), 0.5 mM L-glutamine and 1% penicillin-streptomycin. All neurons were maintained at 37°C in 5% CO₂. Neurons were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. pCAGGS-GRASP65-GFP was transfected at DIV 4. Other constructs were transfected at DIV 2–4.