Trka fc

We established DRG-OPC co-cultures as previously described (19 (link)). DRG were pulled out from spinal cord of E14.5 mouse embryos, put in culture (1 DRG/coverslip) in Neurobasal supplemented with 2% B27 and nerve growth factor (NGF) (100 ng/ml; Harlan, Milan, Italy) and cycled with fluorodeoxyuridine (10 μM; Sigma Aldrich, Milan, Italy) to remove non-neuronal cells. After three weeks, when neurites were well extended radially from DRG, we added 35 × 10³ OPCs to each DRG explant and maintained the explant in MEM (Life Technologies Monza, Italy) supplemented with glucose (4 g/l; Sigma Aldrich, Milan, Italy), 10% FBS and l-glutamine (2 mM; EuroClone, Milan, Italy). To induce myelination, recombinant chimeric tyrosine kinase receptor TrkA Fc (1 μg/ml; Sigma Aldrich, Milan, Italy) was added to the culture medium the following day.

DRG-OPC co-cultures were prepared according to a previously described protocol [31 (link)]. Briefly, DRG from E14.5 mouse embryos were plucked off from spinal cord, put in culture (1 DRG/coverslip) in Neurobasal supplemented with B27 in the presence of nerve growth factor (NGF) (100 ng ml⁻¹; Harlan, Milan, Italy) and cycled with fluorodeoxyuridine (10 μM; Sigma Aldrich, Milan, Italy) to eliminate all non-neuronal cells. After 20 days, when neurites were well extended radially from DRG explants, 35 × 10³ OPCs were added to each DRG in culture and kept in Minimum essential media MEM (Life Technologies Monza, Italy) supplemented with glucose (4 g l⁻¹; Sigma Aldrich, Milan, Italy), 10% FBS and 2 mM l-glutamine (EuroClone, Milan, Italy). Myelination was induced the following day by the addition of recombinant chimeric tyrosine kinase receptor TrkA Fc (1 μg ml⁻¹; Sigma Aldrich, Milan, Italy) to the culture medium.