Anti pdgfrα

Isolation of OLs and astrocytes was performed as described previously (43 (link)). Mice were deeply anesthetized and then forebrains were dissected and minced. Then, the tissues were incubated with PBS containing papain (Worthington, LS003119) and deoxyribonuclease I (Thermo Fisher Scientific, EN0521) for 30 min at 25°C. After digestion, the tissues were triturated using a dounce homogenizer and then filtered through a 40-μm cell strainer. Single-cell suspensions were incubated with anti-PDGFRα (Miltenyi, 130-101-502), anti-O4 (Miltenyi, 130-096-670), or anti-ASCA2 microbeads (Miltenyi, 130-097-679) at 4°C for 4 to 6 hours. Cells bound to the beads were harvested for total RNA or protein extraction.

Primary cultures of OPCs derived from neonatal rats were expanded and reseeded 500,000cells/well in a 48-well plate. Settled cells were cultured with 10 µM EdU for 72h in media containing Neurobrew, N2 and PDGF-BB as described above. In addition, media was supplemented with full (1/1), half (1/2) of absent (0) of bFGF concentration referred to the concentration described above. Cells were harvested with Accutase, labeled with anti-PDGFRα (Milteny Biotec, Bergisch, Germany). Dead cells were excluded using near IR Live/Dead (Thermo Fisher Scientific, Waltham, MA). Samples were analyzed with a 3-laser Beckman Coulter Gallios using Kaluza Software (Beckman Coulter, Brea, CA).