2.4. Primary trigeminal ganglia neuronal cultures

Sensory TG neurons from Sprague–Dawley rats were excised aseptically and placed in Hank buffered salt solution (Life technologies) containing penicillin (100 U/mL) and streptomycin (100 μg/mL, Cat#15140; Life technologies). The ganglia were further dissected to remove all non-neuronal structures before enzymatic dissociation by a 45-minute incubation (37°C) in a DMEM solution containing neutral protease (3.125 mg/mL⁻¹, Cat#LS02104; Worthington, Lakewood, NJ) and collagenase type I (5 mg/mL⁻¹, Cat#{"type":"entrez-nucleotide","attrs":{"text":"LS004194","term_id":"1321650530","term_text":"LS004194"}}LS004194; Worthington). The dissociated cells were resuspended in complete TG medium (ie, DMEM containing penicillin [100 U/mL], streptomycin [100 μg/mL], 30 ng/mL⁻¹ nerve growth factor, and 10% fetal bovine serum [Hyclone, Logan, UT]). For Ca²⁺ imaging (see below), the cells were seeded on poly-d-lysine (Cat#P6407; Sigma) coated glass coverslips (Cat#72196-15; Electron Microscopy Sciences, Hatfield, PA) as a drop of 20 μL on the center of each coverslip, then placed in a 37°C, 5% CO₂ incubator for 45 to 60 minutes to allow cells to attach. Then, the cultures were flooded by gently adding complete TG medium on the edge of each well to avoid detaching any weakly adherent cell. All cells were used within 24 hours after seeding.