SC-DRG Co-Cultures for Myelination Studies

Schwann cell (SC)-dorsal root ganglia (DRG) co-cultures were prepared from either E15.5 rat or E13.5 mouse embryos according to standard procedure (Taveggia and Bolino, 2018 (link)). After DRGs were digested with 0.25% Trypsin (Invitrogen) at 37 °C for 45 min, the reaction was stopped by adding deactivated fetal calf serum (FCS; HyClone) and basic medium (1% Penicillin/ Streptomycin (Lonza), 10% FCS, 50 ng/ml nerve growth factor (NGF; Alomone Labs) in minimum essential medium (MEM; Gibco)) and cells were plated on collagen-coated coverslips. On day 7, myelination was induced by culturing the cells in basic medium with 50 ng/ml ascorbic acid (AA; Sigma). For PTEN inhibition in Pmp22^tg cultures, cells were treated with 1% DMSO (Sigma) as a control or with the PTEN inhibitor VO-OHpic (Rosivatz et al, 2006 (link)) (Sigma) at 50 nM, 500 nM and 5 μM, respectively. In Pmp22^+/- cultures, cells were treated with either 1% DMSO as control, 20 nM mTOR inhibitor Rapamycin (LC Laboratories) or 10 µM PI3K inhibitor LY294002 (Cell Signaling, #9901). Medium was changed every 2–3 days for 2 weeks. HEK293T cells (Sigma) were maintained in DMEM and regularly tested for mycoplasma contamination. Cells were transiently transfected with ALFA-tagged (Gotzke et al, 2019 (link)) PMP22 and Flag-tagged or untagged PTEN expression plasmids (Vectorbuilder) via polyethylenimine.