Dorsal Root Ganglion Dissociation Protocol

Dorsal root ganglion (DRG) from all vertebral levels was harvested and dissociated using methods described previously.^{56 (link),59 (link),88 (link)} Briefly, DRG were dissected from naive animals and treated with collagenase, type I (5 mg/mL), and neutral protease (3.125 mg/mL) in bicarbonate-free DMEM for 45 minutes (Worthington Biochemical, Lakewood, NJ). Mechanical trituration in culture medium (DMEM with 10% fetal bovine serum, penicillin [100 mg/mL], streptomycin [100 U/mL], normocin [0.8 μg/mL], and nerve growth factor [30 ng/mL], Life Technologies, Carlsbad, CA) was then used to dissociate the cells. The cells were then plated in 20 μL of medium on poly-L-lysine/laminin-coated coverslips (BD bioscience, San Jose, CA) and incubated for up to 2 hours before culture media was added to the wells up to a final volume of 1 mL. The cells were then left undisturbed at 37°C (with 5% CO₂) for 12 to 18 hours to allow adhesion.