The experiments designed to isolate single trigeminal ganglion (TG) neurons from humanized wild-type (118AA) and mutant (118GG) mice were approved by the Penn State College of Medicine Institutional Animal Care and Use Committee (Hershey, PA). The mice were first anesthetized with carbon dioxide and then decapitated with a laboratory guillotine. The TG were quickly removed and cleared of connective tissue in ice-cold Hank’s balanced salt solution (Sigma Aldrich, St. Louis, MO). Enzymatic dissociation of the ganglia was performed in modified Earle’s balanced salt solution containing 0.6 mg/ml collagenase (Roche Applied Science, Indianapolis, IN), 0.4 mg/ml trypsin (Worthington Biochemical, Lakewood, NJ) and 0.1 mg/ml DNase (Sigma Aldrich) for 40 min at 35°C in a shaking water bath. Afterwards, the neurons were dispersed by vigorous shaking and centrifuged twice at 130 X g for 6 min. The isolated TG neurons were re-suspended in minimal essential medium, supplemented with 10% fetal calf serum, 1% penicillin-streptomycin and 1% glutamine (all from Invitrogen Corp., Carlsbad CA) and plated onto 35 mm poly-L-lysine coated dishes. The dispersed neurons were then stored in a humidified atmosphere containing 5% CO2/95% air at 37°C.
