CD-1 mice (DRG and spinal cord) or Wnt1-Cre:R26RTomato mice (sympathetic) were sacrificed and tissues of interest collected in freshly oxygenated, ice cold aCSF (see above). Sympathetic (SG) and dorsal root ganglia (DRG) were dissected and dissociated as described before (Furlan et al., 2016), with minor modifications. Briefly, following dissection (DRG: ∼30 ganglia collected in total from Cervical1-Lumbar6; SG: thoracic1-12 and stellate), the ganglia got transferred into a 3cm plastic dish with 2.7ml of pre heated (37°C) digestion solution (400μl TrypLE Express (Life Technologies), 2000μl Papain (Worthington; 25U/ml in aCSF), 100μl DNase I (Worthington; 1mM in aCSF) and 200μl Collagenase/Dispase (Roche; 20mg/ml in CS)). Non-ganglia tissue was removed from the ganglia. After 30 min incubation at 37°C, ganglia were triturated with 0.5% BSA-coated glass Pasteur pipette (flamed to 70% of original opening). DRG were also carefully ripped open by using fine forceps to make cells more accessible for the enzymes. This procedure was repeated every 20-30 min using Pasteur pipettes with decreasing diameter appropriate to the dissociation state. Depending on the dissociation progress 50μl of Collagenase/Dispase (20mg/ml) and 100μl of TrypLE solution was added.
