Ng2 af488

Mouse OPCs were generated from epiblast stem cells (EpiSCs) as previously described except SHH was not used for maintenance or differentiation of OPCs (Najm et al., 2011 (link)). Mouse protocols were approved by Case Western Reserve University School of Medicine’s Institutional Animal Care and Use Committee (IACUC). In brief, epiblast stem cells (EpiSCs) were isolated from 129S/SvEv male embryos (E3.5; The Jackson Laboratory), pushed to form neural rosettes, and then rosettes were passaged into nunclon plates coated with poly-L-ornithine (Sigma, P3655–50MG) and laminin (Sigma, L2020–1MG) in OPC growth media consisting of DMEM/F12 supplemented with N2 Max (R&D Systems, AR009), B27 (Thermo Fisher, 12587010), 20ng/mL bFGF (R&D Systems, 23–3FB-01M), and 20ng/mL PDGFA (R&D Systems, 221-AA). Media was changed every 48 hours and cultures were maintained in 37°C with 5% CO₂. After 4 passages these EpiSC derived OPCs were sorted to purity by fluorescence activated cell sorting using conjugated CD140a-APC (eBioscience, 17-1401-81; 1:80) and NG2-AF488 (Millipore, AB5320A4; 1:100) antibodies.

OPCs used in this study were generated from two separate EpiSC lines, EpiSC9 (female) and 129O1 (male), using in vitro differentiation protocols and culture conditions described previously^{3 (link),23} . Cultures were regularly tested and shown to be mycoplasma free. To ensure uniformity throughout all in vitro screening experiments, EpiSC-derived OPCs were sorted to purity by fluorescent activated cell sorting at passage five with conjugated CD140a-APC (eBioscience, 17–1401; 1:80) and NG2-AF488 (Millipore, AB5320A4; 1:100) antibodies. Sorted batches of OPCs were expanded and frozen down in aliquots. OPCs were thawed into growth conditions for one passage prior to use in screening assays.