Nrg1β1

To harvest Schwann cells (SC), rat sciatic nerves were exposed, removed, and kept in DMEM plus glutamax (Invitrogen, UK) containing 100 U/mL penicillin and 100 μg/mL streptomycin. Nerves were then dissected in trunks, desheathed, and finally chopped in 1 mm segments. The segments were then plated in a Petri dish in SC growth medium (DMEM plus glutamax containing 100 U/mL penicillin, 100 μg/mL streptomycin, 14 μM forskolin, and 100 ng/mL NRG1β1, R&D Systems, UK). Cells were incubated for 2 weeks at 37°C with fresh medium added approximately every 72 h. After these 2 weeks medium was aspirated and 0.125% (w/v) collagenase type IV and 117 U/mg dispase were added to the Petri dish. After 24 hours (h) incubation, cell suspension was filtered through a 70 mm cell strainer (Falcon; BD Biosciences Discovery Labware, Bedford, MA) and centrifuged at 100 ×g for 5 min to obtain the cell pellet. Finally, the cell pellet was resuspended in SC growth medium, seeded into a Petri dish pre-coated with poly-D-lysine (Sigma, St Louis, MO, USA), and incubated in the same conditions. The following day, the medium was changed and cells were left to proliferate. When confluent, SC were purified by an antibody complement method to eradicate the remaining fibroblasts [14 (link)–16 (link)].