Collagenase dispase

Dorsal root ganglia (DRG) were resected from 6-week-old Swiss Webster mice (Hilltop Laboratories, Scottdale, PA, USA) and enzymatically digested using papain and collagenase/dispase (Worthington Biochemical, Lakewood, NJ, USA), followed by mechanical separation into single-cell suspensions. Neurons were counted and plated on Matrigel-coated (Corning, Silicon Valley, CA, USA) cell culture plates at 3000–80,000 neurons per well, depending on the assay [9 (link)]. DRG neurons were maintained in Neurobasal A medium (Thermo Fisher Scientific, Waltham, MA, USA) with 1% penicillin-streptomycin (PS), 1× Glutamax, DNA synthesis inhibitor 5-fluoro-2′deoxyuridine (FUDR) to deplete non-neuronal cells by mitotic inhibition, and neurotrophic factors (nerve growth factor, glial cell-derived neurotrophic factor, neurturin; obtained from PeproTech, Cranbury, NJ, USA) [9 (link)]. DRGs were allowed to acclimate to the culture plate for 3–4 days before experimental procedures. All studies were conducted in accordance with the Institutional Animal Care and Use Committee at Virginia Tech (protocol approved 8 February 2019).