Isolation and Culture of Adult DRG Neurons

DRG from adult BALB/c mice were excised and cultured as previously described^{7 (link)}. Briefly, DRG from cervical to sacral (up to S2) level were bilaterally excised, collected and accurately de-sheathing in a dish containing cold F12 (Nutrient Mixture F12 Ham) medium (Sigma–Aldrich Inc., Italy). After incubation at 37 °C (1 h) with collagenase from Clostridium hystoliticum 0.125% (Sigma–Aldrich Inc., Italy), DRG were dissociated. Cells were plated on laminin (Sigma–Aldrich Inc., Italy) coated glass coverslips (24 mm) and cultured at 37 °C with 5% CO₂ for 48 h in Bottenstein and Sato medium (BS)^{7 (link)} supplemented with Recombinant Human β-NGF, Recombinant Murine GDNF and Recombinant Human NT3 (Peprotech, Rocky Hill, NJ, USA). The administration of OHP (0.1 μg/ml) and 5-FU (500 nM) was performed 48 h after the isolation of DRG neurons and all the experiments were performed from 48 to 54 h of culture.