DRG Neuron Isolation and Culture

Culture preparation was conducted as previously described (Masuoka et al., 2015 (link), 2016 (link)). The C57BL/6J male and female mice (6- to 14-day-old) were anesthetized by the inhalation of isoflurane (Escain®; Mylan Inc., Cecil Township, PA, USA). DRG were rapidly dissected in ice-cold Ca²⁺/Mg²⁺-free artificial cerebrospinal fluid (Ca²⁺/Mg²⁺-free ACSF; 143.9 mM NaCl, 3.35 mM KCl, 21 mM NaHCO₃, 9.9 mM glucose, 0.6 mM NaH₂PO₄) gassed with a mixture of 95% O₂ and 5% CO₂ (pH 7.4). Neurons were dissociated following treatment with 0.1% type II collagenase (240–265 U/mg; Worthington Biochemical Co., Lakewood, NJ, USA), 0.1% trypsin (Gibco, San Diego, CA, USA), and 0.01% DNase I (Sigma, St. Louis, MO, USA) in Ca²⁺/Mg²⁺-free ACSF and shaken (35 cycle/min) in a water bath at 37°C for 30 min. Cells were gently triturated in Dulbecco's modified Eagle medium (Sigma) containing 10% horse serum (Gibco), 5% fetal calf serum (Gibco), and 1% penicillin–streptomycin (Wako, Osaka, Japan). Dispersed cells were passed through a 100-μm cell strainer (BD Biosciences, San Jose, CA, USA), and the filtered cells were seeded on glass coverslips (13 mm in diameter) coated with poly-L-lysine (Matsunami Glass Ind., Osaka, Japan).