Cell Culture and Differentiation Protocols

PC12 cells were maintained and differentiated with NGF as previously described [18 (link)]. For NGF treatment, cells were grown in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 1% heat-inactivated horse serum (Sigma-Aldrich), penicillin/streptomycin (Thermo Fisher Scientific) and 50 ng/ml recombinant human β-NGF (Alomone Labs, Jerusalem, Israel) for 7-8 days in a 7,5% CO₂ atmosphere at 37°C. Medium was changed every other day and before transfection.
HEK293 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (all from Thermo Fisher Scientific) in a 5% CO₂ atmosphere at 37°C.
Rat primary cortical cultures were prepared as previously reported [19 (link)]. Briefly, cortex from embryonic (E18) Sprague-Dawley rats were dissected out, dissociated and plated at a density of 250 cells/mm² on poly-L-lysine-coated (Sigma-Aldrich) coverslips or at a density of 700 cells/mm² on poly-L-lysine-coated plates. Neurons were maintained in neurobasal medium with B27 and 2mM GlutaMAX (all from Gibco). However, neurons plated on coverslips were initially seeded with MEM medium supplemented with 100 mM pyruvic acid (Gibco), 20% glucose (Sigma-Aldrich) and 10% heat-inactivated horse serum.