Cell cultures and treatments

BV-2 murine microglia cell was routinely grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS, Gibco), penicillin (0.1 %), and streptomycin (0.1 %) at 5 % CO₂, 37 °C.

BV-2 microglia cells were treated according to the method described with modifications [23]. Briefly, 96-well plates (Corning, New York, USA) were coated with methanol-solubilized nitrocellulose and washed with ddH₂O. Then, the wells were incubated with poly-L-lysine (PLL, 0.05 mg/ml, Sigma, St Louis, MO, USA) for 2 h at 37 °C and washed with ddH₂O. Later, Nogo-P4 (Alpha Diagnostic International Inc., San Antonio, TX) was applied at 100 μg/mL and coated overnight at 4 °C. Nogo-P4 is a 25 aa inhibitory peptide sequence (residues 31–55 of Nogo-66), a potent inhibitory component of Nogo-A [3]. After transfection, BV-2 microglia cells were added onto the wells pre-coated with Nogo-P4 or PBS at a density of 5 × 10⁵ cells/mL and cultured for 6 h. Then, conditioned medium was collected and centrifuged to discard cell debris for further experiment.

Primary cortical neuron cultures were prepared following the described method with some modifications [24]. Briefly, cortices of C57BL/6J mouse at embryonic 15–17 days were removed, dissected free of meninges, and dissociated in 0.125 % trypsin. Then, dissociated cells were plated in PLL (0.05 mg/ml, Sigma)-coated 24- or 96-well plates at a density of 3 × 10⁵ cells/mL in DMEM supplemented with 10 % FBS and cultured at 37 °C in 5 % CO₂. After 4 h, the medium was replaced with neurobasal medium (Gibco) supplemented with B27 supplements (Gibco). About 95 % of these cells were positive for microtubule-associated protein 2 (MAP2), a marker for neurons. After 6 days of in vitro culture, the medium was replaced with BV-2 microglia-conditioned medium for a further 24 h.