Isolation and Polarization of Rat Microglia

Primary microglia were isolated from adult male Sprague-Dawley rat brains, as described previously [25 (link)]. The cells were cultured (1.2 x 10⁵ cells per well in 2 mL of medium) in six-well poly-d-lysine-coated plates (Sigma-Aldrich, P0296) and grown in microglia culture medium (Dulbecco's modified Eagle's medium/F-12 Glutamax; Gibco, 10565042) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin and 10 ng/mL rat recombinant carrier-free macrophage colony stimulating factor (M-CSF; Peprotech, 400-28) at 37°C with 5% CO₂. Half of the medium was changed every three days. M0, M1 and M2 microglia were polarized as described previously [26 (link)].