Culturing HEK-293T and DRG Neurons for Viral Transduction

HEK-293T cell cultures and DRG neuronal cultures were prepared according to previously described methods (10 (link), 32 (link)). Briefly, HEK-293T cells were cultured in Dulbecco’s modified Eagle’s medium/high glucose medium (Gibco/Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) and 1% antibiotics. DRGs from adult mice were collected in ice-cold Neurobasal medium (Life Technologies) with 10% FBS (J R Scientific) and penicillin (100 U/ml) and streptomycin (100 µg/ml) (Quality Biological) and then digested with enzyme solution [dispase (5 mg/ml) and collagenase type I (1 mg/ml)] in Hanks’ balanced salt solution without Ca²⁺ and Mg²⁺ (Life Technologies) at 37°C for 20 to 25 min. After trituration and centrifugation, the dissociated neurons were resuspended in mixed Neurobasal medium and plated into six-well plates coated with poly-d-lysine (50 µg/ml) (Sigma) at a density of 1.5 × 10⁵ to 4 × 10⁵ cells. The neurons were incubated in a humidified 95% O₂ and 5% CO₂ atmosphere at 37°C. For viral infection, 2 µl of AAV5 virus (titer ≥ 1 × 10¹²/ml) was added to each 2-ml well after 24 hours of incubation. For siRNA transfection, siRNA was diluted to the concentration of 100 nM and transfected to cultured cells by Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instruction. Cells were collected 2 or 3 days later for Western blot analysis.