C6645

Organotypic hippocampal slice cultures were prepared from P5 C57BL/6 wild-type mice of both sex according to.³⁷ (link) In brief, mice were decapitated and the hippocampus was extracted and sliced into coronal sections of 300 μm thickness. The slices were placed on small pieces of Polytetrafluoroethylene (PTFE) membrane, pore size 0.45 μm (Millipore, # FHLC01300) and cultured on cell culture insert of 0.4 μm pore size (Millipore, # PICM03050) placed in a 6-well plate and then incubated at 37°C with 5% CO₂ for 18 to 23 days in vitro (DIV). The inhibitor mix containing Ara-C (Sigma-Aldrich, #C6645), Uridine (Sigma-Aldrich, #U3750) and 5-Fluoro-2′-desoxyuridine (Sigma-Aldrich #F0503), was added to a final concentration of 3 μM on the third day and the medium was exchanged three times per week.

SCs were cultured and purified as previously described (Gu et al. 2014 (link)). Sciatic nerves were harvested from neonatal rats (1–2 days), cut into small pieces and enzymatically treated with 1% collagenase (17018-029, Gibco, Carlsbad, CA, USA) and 0.125% trypsin (25200-056, Gibco). The cell mixture was then resuspended in DMEM (11965-092, Gibco), 10% FBS (12483-020, Gibco), and penicillin–streptomycin (PS; 15140122, Thermo Fisher Scientific, Cleveland, OH, USA) using a 400-mesh cell screen to make a single-cell suspension, seeded in 50 μg/ml poly-d-lysine (PDL; P-7890, Sigma, St Louis, MO, USA) coated dishes with 10 mM cytosine arabinoside (C6645, Sigma) and 10% FBS in DMEM for 1 day to remove fibroblasts. Afterwards, the medium was changed to supplemented with 5 μM forskolin (F6886, Sigma), 2 ng/ml Neuregulin 1 (Nrg1; 396-HB-050, R&D, MN, USA) and 10% FBS in DMEM. After 48 h, cells were incubated with anti-Thy1 antibody (1:1000, M7898, Sigma) to remove remaining contaminating fibroblasts. SC purity was assessed by S100β (1:500, S2532, Sigma) immunostaining. Passage 2 SCs with > 95% purity were used for all cell experiments.