The potentiality of EGF/bFGF-responsive aDRG and eDRG NSCs was characterized by differentiation. The EGF + bFGF supplementation was withdrawn and neurospheres were subplated into 16-well chamber slides (Nunc, Rochester, NY) coated with poly-D-lysine (20 mg/ml, Sigma) and laminin (1 mg/ml, Sigma). Cells differentiated for 7–21 days. Neurobasal medium supplemented with 1.2% penicillin-streptomycin (Invitrogen) for differentiation induction (2% fetal bovine serum, FBS) for 2 days and was followed by maintenance medium (10% FBS) for the remainder of the experiment. DRG NSCs cultures differentiated in a humidified incubator at 37°C; medium was changed twice per week. At the end of the differentiation period, the cells were washed with 0.1 M PBS and fixed with 4% formaldehyde for immunocytochemistry. Fixation for VGluT2 immunostaining included the use of freshly made 4% formaldehyde + 0.4% picric acid.
