Isolation of Dorsal Root Ganglia Neurons

Dorsal root ganglia from all levels were acutely dissociated from 100 g Sprague-Dawley rats and DRG neurons were isolated as we described previously [13 (link),39 (link),24 (link),42 (link),35 (link),38 (link),43 (link)–45 (link),22 (link),46 (link)]. In brief, removing dorsal skin and muscle and cutting the vertebral bone processes parallel to the dissection stage-exposed DRG. Dorsal root ganglia were then collected, trimmed at their roots, and enzymatically digested in 3 mL bicarbonate-free, serum-free, sterile DMEM (Cat# 11965, Thermo Fisher Scientific) solution containing neutral protease (3.125 mg.ml⁻¹, Cat# LS02104; Worthington, Lakewood, NJ) and collagenase type I (5 mg.ml⁻¹, Cat# LS004194, Worthington, Lakewood, NJ) and incubated for 60 minutes at 37°C under gentle agitation. Dissociated DRG neurons (~1.5 × 10⁶) were then gently centrifuged to collect cells and washed with DRG media DMEM containing 1% penicillin/streptomycin sulfate from 10,000 μg/mL stock, 30 ng/mL nerve growth factor, and 10% fetal bovine serum (Hyclone) before plating onto poly-D-lysine- and laminin-coated glass 12- or 15-mm coverslips. All cultures were used within 48 hours.