Isolation and Culture of Mouse Oligodendrocyte Precursor Cells

Mouse OPCs were isolated from P6–P9 pups, as described previously (Watkins et al., 2008 (link); Swire and ffrench-Constant, 2019 (link)). Ear clips were taken for subsequent genotyping. Briefly, cerebral cortices were dissected, diced, and dissociated into single-cell suspensions gently using MACS Neural Tissue Dissociation Kit P (catalog #130–092-628, Miltenyi Biotec). Cells were resuspended in 0.2% BSA, insulin, and PBS, and were transferred to treated tissue culture dishes coated with BSL1 (catalog #L-1100, Vector Laboratories) twice for 15 min. Cell solutions were then transferred to dishes coated with anti-PDGFRα (CD140a) for 45 min. Solutions were aspirated, and attached cells were washed twice with media and removed with a cell scraper. All collected cells were added to vented T75 flasks and grown at 37°C with 7.5% CO₂. Cells were grown in myelination media containing PDGF and neurotrophin-3, were changed every 2 d, and were supplemented daily with PDGF. After 7–9 d, confluent flasks were washed with PBS and then detached using TrypLE for 10 min at 37°C. Solutions were centrifuged at 1000 rpm for 5 min, resuspended, and counted using a hemocytometer.