Isolation and Purification of Adult Microglia

Adult microglia were isolated based on a modified version of an established protocol^{89 (link)} LH rats were deeply anaesthetised with sodium pentobarbital (Euthatal^®, 80 mg/kg, administered intraperitoneally) and transcardially perfused with ice-cold DPBS containing 2 mM EDTA. Brains were dissected, homogenised with Dounce homogeniser in 5 ml ice-cold DPBS, filtered through a 70 µm cell strainer to remove any debris and centrifuged (300 × g, 7 min, 4 °C). Pellets were resuspended in 7 ml of 30% Percoll^® (Sigma, P1644) and centrifuged (800×g, 30 min, room temperature), to remove myelin. Microglia were isolated from the pellet with CD11b conjugated magnetic microbeads (Miltenyi Biotec) according to manufacturer’s protocol. Magnetic-activated cell sorted CD11b+ cells were flushed from LS columns (Miltenyi Biotech) and collected in complete medium DMEM/F12 supplemented with 10% FBS and Pen-Strep. Cells were seeded at 100,000 cells/cm² concentration in 24-well plates coated with poly-d-lysine (0.1 mg/ml. Sigma-Aldrich).