Ham s nutrient f12 culture medium

DRG neurons’ cultures were prepared by collecting DRGs from the first cervical to the sixth lumbar segment into Ham’s nutrient F12 culture medium (Sigma) supplemented with 2% Ultroser G (Pall SA, Saint-Germain-en-Laye, France), 1 mM glutamine (Invitrogen, Waltham, USA), 50 IU/ml penicillin (Invitrogen, Waltham, USA) and 50 μg/ml streptomycin (Invitrogen, Waltham, USA). Following incubation in 2000 U/ml collagenase type IV (Worthington Biochemical Corp., Lakewood, USA) for 3 h, DRG were triturated, and the cells were plated onto poly-DL-ornithine (Sigma)-coated glass coverslips. Cells were grown for 24 h, at 37 °C in the supplemented medium, to which the nerve growth factor (NGF, 50 ng/mL; Promega, Southampton, UK) was added.

Dorsal root ganglia from the C1 to the S1 segment were dissected and placed into Ham’s nutrient F12 culture medium (Sigma, UK) supplemented with 1 mM L-glutamine (Invitrogen, UK), 5000 IU/ml penicillin (Invitrogen, UK), 5000 μg/ml streptomycin (Invitrogen, UK) and 2% ultroser G (Biospectra, France). Ganglia were incubated in type IV collagenase (Lorne Diagnostics, UK, 300 U/ml) for 3 hours at 37 °C at 5% CO₂. Following several washes in the supplemented culture medium, ganglia were triturated with fire-polished Pasteur pipettes, and plated on poly-DL-ornithine (Sigma, UK)-coated glass coverslips in the supplemented medium. Cells were grown in the supplemented medium in the presence of 50 ng/ml nerve growth factor (NGF, Promega, USA) for16–72 hours.