Primary Schwann cell culture
Primary Schwann cells were prepared from 4-month-old C57BL/6 mice according to Manent's protocol [32] with minor modifications. Briefly, sciatic nerves were removed and incubated for 7 days in the pre-treatment medium, which consisted of D-MEM (high glucose) supplemented with 10% FCS, 50 mg/ml gentamicin (Invitrogen Corp.), 2.5 μg/ml fungizone (Invitrogen Corp.), 2 μM forskolin (EMD Biosciences Inc.) and 10 ng/ml recombinant heregulin-beta1 (R&D Systems). For dissociation, cultured sciatic nerves were cut into pieces and incubated at 37°C for 3 hours in Opti-MEM medium (Invitrogen Corp.) containing 130 U/ml collagenase type I (Invitrogen Corp.) and 0.4 U/ml dispase II (Roche Diagnostics). Dissociated cells were resuspended in the pre-treatment medium and plated on Poly-D-Lysine/Laminin coated plate (BD Biosciences). The purity of the cultured Schwann cells, as determined by indirect immunofluorescence analysis, approached 90 %.