Bme f12

Purified OPCs were placed on coverslips coated with poly-L-lysine (Sigma) and laminin (Engelbreth-Holm-Swarm murine sarcoma; Sigma; 2 × 10⁴ cells/well) and they were cultured with dual PDE7-GSK3 or GSK3 inhibitors (VP3.15 or TDZD8; 1 μM) in a previously described serum-free differentiation medium78 (link), consisting of BME:F12 (1:1; Invitrogen) supplemented with 100 μg/ml transferring (Sigma), 20 μg/ml putrescine (Sigma), 12.8 ng/ml progesterone (Sigma), 10.4 ng/ml sodium selenite (Sigma), 25 μg/ml insulin (Sigma), 0.8 μg/ml thyroxine (Sigma), 0.6% glucose (Normapur), 6.6 mM glutamine (Invitrogen) and 100 U/mL penicillin, 0.1 mg/ml streptomycin and 0.25 μg/ml Amphotericin B antibiotic anti-mycotic solution (Sigma). After 2 DIV, they were fixed with 4% paraformaldehyde (PFA) and subjected to immunocytochemistry for active caspase 3 (Casp3; 1:200; Abcam; Cat#ab13847) and the OPC marker A2B5 (1:10; Hybridoma Bank). 10–20 microphotographs from each coverslip were taken randomly with an In Cell Analyzer 1000 (GE-HealthCare) and the number of Casp3⁺-A2B5⁺ double positive cells was counted using the software In Cell Analyzer 1000 Workstation (GE-HealthCare). Data were expressed as a mean ratio of double positive cells for Casp3 and A2B5 with respect to the total number of A2B5⁺ cells ± SEM for each condition in at least three independent experiments.

Brain slices were obtained from E12.5 wild-type mice and the CH and the PSB from E12.5 mTmG mouse embryos as described [see also (Bribian et al., 2014 (link))]. Slices were transferred to collagen-coated culture membrane (PICM0RG50, Millipore) in 1.2 ml of medium BME-F12 1:1 (41010-026, Invitrogen), glutamine (25030-024, Invitrogen), 5% horse serum (26050-088; Invitrogen), penicillin, streptomycin (15140122, Invitrogen), and 5% bovine calf serum (12133C, Sigma-Aldrich). The CH and PSB from wild-type slices were removed and replaced with the dissected CH or the PSB from mTmG mice slices. After several hours, the medium was changed to Neurobasal™ medium supplemented as above and cultured for up to 48 h before analysis.