Primary culture of trigeminal ganglia neurons

Trigeminal ganglia (TG; n = 3–4 rats per 24-well plate run in triplicate) were extracted from adult OVX female rats (~200 g) immediately following decapitation. Primary neuron cultures were prepared using previously described methods (Loyd et al., 2011, Patwardhan et al., 2005). Briefly, TGs were suspended in HBSS on ice and gently washed three times. After dissociation with collagenase (5%, Worthington Biochemical Corp, Lakewood, NJ) and trypsin (1%, Sigma–Aldrich) at 37 °C, the cells were suspended in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Waltham, MA) containing 10% fetal bovine serum, glutamine, penicillin–streptomycin, nerve growth factor (NGF, 100 ng/ml; Harlan, Indianapolis, IN), and treated with mitotic inhibitors (5-fluoro-2′-deoxyuridine and uridine). Cells were then lightly dissociated using a 20-gauge followed by a 23-gauge needle and then applied to 24-well poly-D-lysine-coated plates (Corning Inc., Corning, NY) and maintained in an incubator at 37 °C and 5% CO₂.