Isolation and Culturing of Microglia and OPCs

Sprague Dawley cortices were digested and grown in DMEM containing 10% FBS with 4.5 g/l glucose, L-glutamine, pyruvate, and 1% penicillin/streptomycin (DMEM-10%) using standard methods [31 (link)]. After 7–10 days, microglia and OPCs were purified by differential attachment [32 (link)]. Microglia were plated in DMEM-10% on poly-L-lysine (PLL, Sigma, 13 µg/ml) coated glass coverslips (VWR, 10 µg/ml) at 5 × 10⁴ cells/coverslip. Microglia were stained using anti-mouse CD4 (1:100, ThermoFisher Scientific) and anti-rabbit Iba-1 (1:100, Wako) using standard immunohistochemistry methods described above. OPCs were plated onto 13 mm PLL coated glass coverslips at 4 × 10⁴ cells/coverslip in DMEM-BS containing FGF2 (50 ng/mL) and PDGF (50 ng/mL) for 5 days, then treated in duplicate with IL-16 (100 ng/ml) or DMEM-BS for 4 days. On day 5, cells were immunolabelled with anti-rabbit NG2 (1:100, Abcam), anti O4 (1:100, IgM, hybridoma) or anti-rat PLP (1:100, AA3, hybridoma) and 10 images taken per coverslip with 2 coverslips per treatment, with an average of 200–300 cells/coverslips analysed. OPCs were stained using CD4 described above and co-stained using anti-O4 (1:100, IgM, hybridoma).