Mouse Dorsal Root Ganglion Neuron Isolation

Mouse DRG neurons were extracted and dissociated as previously described [55 (link)]. Briefly, the mice were euthanized by cervical dislocation. The spinal cord was removed and 20–40 DRGs were dissected and washed in cold HBSS solution (Thermo Fisher Scientific). For IHC, whole ganglia were directly fixed for 2 h in 4 % PFA. For ICC, ganglia were then incubated in 900 U/mL type XI collagenase (Sigma-Aldrich) and 5.46 U/mL dispase (Thermo Fisher Scientific) for 45 min at 37 °C in 5% CO₂. After enzymatic treatment, the ganglia were mechanically dissociated using fire-polished glass pipettes in calcium-free solution containing HBSS (Thermo Fisher Scientific), 1% MEM-Vit (Thermo Fisher Scientific), 10% fetal bovine serum (Thermo Fisher Scientific) and 100 mg/mL penicillin/streptomycin. The cell suspension was centrifuged, and the pellet was resuspended in culture medium containing: MEM (Thermo Fisher Scientific), 1% MEM-Vit (Thermo Fisher Scientific), 10% fetal bovine serum (Thermo Fisher Scientific) and 100 mg/mL penicillin/streptomycin. The cells were then seeded onto 6 mm diameter glass coverslips previously coated with 0.01% poly-L-lysine (Sigma-Aldrich, St. Louis). Twenty-four hours after seeding, the cells were subjected to calcium imaging or fixed for 10 min in 4%PFA.