Isolation and Culture of Mice DRG Neurons

Mice at the age of 12–16 weeks were sacrificed with CO₂, and their DRG neurons were collected and cultured in accordance with the procedures described in Cheng et al., (2010) [14 (link)] and Lin et al., (2016) [15 (link)]. The dissected DRGs were digested with 0.125% collagenase (type I, Sigma-Aldrich, St. Louis, MO, USA) and 2 units/mL dispase II (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C. The segregated neurons were triturated using a flame-polished Pasteur pipette and seeded on laminin (Sigma-Aldrich, St. Louis, MO, USA)-coated polydimethylsiloxane (PDMS, UNI WARD, New Taipei City, Taiwan) substrate, which was prepared on a 12 mm coverslip with a base-to-curing-agent ratio of 35:1. Before the DRG neurons were seeded, the PDMS-covered coverslips were exposed to ultraviolet light for 15 min and coated with poly-L-lysine (0.01%, Sigma-Aldrich, St. Louis, MO, USA) for 10 min; then, they were coated with laminin (10 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) for 2 h. The neurons were cultured in a 3.5 cm Petri dish with DMEM plus 10% fetal bovine serum and maintained in an incubator with 5% CO₂ at 37 °C for 2–3 days. The cultured DRG neurons were then subjected to electrophysiological recordings of acid-induced currents or mechanically activated currents.