Isolation and Culture of Mouse Dorsal Root Ganglia

For each tissue preparation, mice were euthanized via induction of isoflurane briefly followed by decapitation. The spinal column was removed and bisected; lumbar DRG were removed and pooled, and subjected to enzymatic incubations as described [20 (link),57 ,84 ]. DRG tissue was incubated in papain (45 U in HBSS+H, Worthington) for 20 min at 37°C, rinsed, and incubated in collagenase (1.5 mg/mL in HBSS+H, Sigma-Aldrich) for 20 min. DRG were washed again with HBSS+H, then transferred to 1mL of DRG media, which consisted of Neurobasal A medium (Invitrogen) supplemented with 100 U/mL penicillin/streptomycin (Corning), 2 mM GlutaMAX (Life Technologies), 2% B27 (Gibco) and 5% fetal bovine serum (Gibco). DRG were then manually triturated with fire-polished Pasteur pipettes (VWR) and passed through a 40-μm filter (VWR). DRG were then plated onto poly-d-lysine/collagen (Sigma-Aldrich)-coated 12-mm glass coverslips (Thermo Fisher Scientific) and stored at 37°C and 5% CO₂ until testing.