All culture conditions were initially tested with mouse primary microglia. C57Bl/6 neonates were cold/CO2 anesthetized in a dry ice chamber following approved protocols and whole brains were harvested. After meningeal removal, cortical cups were isolated, minced on ice, and crudely triturated with a wide-bore fire-polished pipette in a solution of Accutase (Life). The preparation was incubated at 37°C with rotation for 10′, followed by further trituration with a narrowed pipette. The suspension was further incubated for 10′ at 37°C with DNase I 0.1%. The final trituration was left to settle for 1 minute, and the supernatant was filtered through a 70μm mesh, and spun through a 4% BSA cushion to remove cell debris. This preparation was plated directly on matrigel coated plate, and grown in complete Neurobasal with 5% serum. After a week, loosely adherent microglia were harvested and further used on different surfaces. Alternatively, MACS sorting (Miltenyi) for CD11b was applied to the cortical suspension, following the manufacturer’s instructions. Flow-through was discarded while retained cells were eluted directly onto test surfaces and used accordingly.
