Dorsal root ganglia (DRG) and trigeminal ganglia (TG) dissection and dissociation

Male or female adult mice were anesthetized by CO₂ inhalation and decapitated before dissection. The spinal column was isolated and a laminectomy was performed to expose the dorsal side of the spinal cord. The spinal cord was removed by cutting attached dorsal roots. DRG were located by following dorsal root branches to intervertebral foramina, carefully dissected and placed in chilled Hanks balanced salt solution. TG were located at the base of the skull following removal of the brain and were carefully removed and placed in chilled Hanks balanced salt solution. To dissociate into individual neurons, DRG or TG were transferred to a enzymatic solution consisting of 1.3 mg/mL collagenase (CLS4; Worthington Biochemical), 0.2 mg/mL trypsin (Worthington Biochemical) and 0.1 mg/mL DNase I in Earles’ balanced salt solution supplemented with 3.6 g/L D-glucose and 10 mM HEPES. Ganglia were incubated at 36 °C for 1 h in a water bath shaker rotating at 110 rpm. After incubation, neurons were mechanically dissociated by vigorously shaking the flask for 10 s. Neurons were centrifuged (60× g for 6 min) and resuspended in minimum essential medium supplemented with 10% fetal bovine serum and 1% antibiotics (MEM+/+, all from Invitrogen) twice before being plated on polyethylenimine (PEI)-coated tissue culture dishes. Cells were maintained in a humidified 95% air/5% CO₂ incubator at 37 °C before experiments.