Isolating and Culturing DRG Neurons

To isolate and culture DRG^{56 (link),57 (link)}, vertebral columns were dissected from euthanized mice, and and lumbar DRGs were isolated. The ganglia were digested with a 0.2 mg/ml collagenase type II solution (Worthington) for 45 min at 37 °C followed by a 20 min incubation after the addition of 2% trypsin. DRGs were mechanically dissociated using a 1 ml syringe by aspirating and ejecting the digested DRG solution ten times with decreasing needle size from 18 gauge to 28 gauge. The cell solution was centrifuged for 5 min at 160 × g, and DRG neurons were resuspended in Neurobasal complete medium supplemented with 5% horse serum and 50 ng/ml β-nerve growth factor (Thermo Fisher Scientific). The cells were then seeded on poly-L-lysine hydrobromide (Sigma-Aldrich, St. Louis, MO, USA) treated glass cover slides and incubated for 1–3 days in a humidified incubator (5% CO₂/95% air) at 37 °C before analysis.