Purcol bovine collagen

DRGs were dissected from embryonic day (E)13.5 mouse embryos and seeded in 12-well plastic (Grenier) or four-well glass-bottom dishes (CellVis) sequentially coated with 1 mg/ml poly-d-lysine (Sigma-Aldrich), 10 μg/ml laminin-entactin complex (Corning), and 0.1 mg/ml PurCol bovine collagen (Advanced Biomatrix). Explants were grown in phenol-red Neurobasal media (Invitrogen) supplemented with 2% B27 serum-free supplement (Invitrogen), 1% l-glutamine (Wisent), 1% penicillin/streptomycin (Wisent), 10 μm 5-fluoro-2’-deoxyuridine (FDU; Sigma-Aldrich), and 12.5 ng/ml NGF (CedarLane) or 37.5 ng/ml BDNF (CedarLane) at 37°C, 5% CO₂. Deprivation of neurotrophic support was accomplished using 2.0 μg/ml of function blocking antibodies against NGF (homemade rabbit polyclonal antibody raised against 2.5s NGF; Acheson et al., 1991 (link)) or BDNF (mouse monoclonal, DSHB #9-b) in complete fresh media without neurotrophic supplementation.

DRG explants were dissected from pregnant CD1 mice with litters of embryonic day E13.5 embryos (Charles River). Explants were seeded on six-well plastic cell culture plates (Greiner) or four-well glass-bottom imaging dishes (CellVis) coated in a three-step process with 1 mg/ml poly-D-lysine (Sigma-Aldrich), 10 μg/ml laminin-entactin complex (Corning), and PurCol bovine collagen 0.1 mg/ml (Advanced Biomatrix). Explants were cultured in neurobasal medium (Invitrogen) supplemented with 2% B₂₇ serum-free supplement (Invitrogen), 1% L-glutamine (Wisent), 1% penicillin/streptomycin (Wisent), and 10 μM 5-fluoro-2’-deoxyuridine (FDU; Sigma-Aldrich) with 12.5 ng/ml NGF (CedarLane). NGF deprivation was achieved using fresh media as described above but lacking NGF and containing 2.8 μg/ml rabbit anti-NGF antibody (produced in-house). A full description of the anti-NGF antibody (raised against 2.5s NGF), its specificity and its biological validation in survival and cell-surface binding assays has been published (Murphy et al., 1993 (link)). All experimental procedures were approved by the Montreal Neurologic Institute Animal Care Committee and University of British Columbia animal care committees and were in compliance with Canadian Council on Animal Care guidelines.