One- or 2-d-old pups were genotyped and their forebrains were dissected in dissection buffer (5% FBS in 1× PBS). The brains were then minced with sterilized razor blades and filtered through a sterile 40-μm filter. Cell suspensions were then pelleted by centrifuging for 5 min at 300 × g. The cells were then resuspended in 15 ml of culture medium (DMEM + 10% FBS + 1% pen/strep) and plated in a 75-mm2 flask. Two days after plating, M-CSF was added to the culture medium at 5 ng/ml. The culture medium was replaced every 2 d, and cells were allowed to grow for 12–14 d postseeding. To harvest microglia, flasks were shaken at 125 rpm for 4–5 h at 37°C. Detached microglia were then collected, pelleted by centrifuging for 5 min at 300 × g. Cells were resuspended in culture medium, counted, and plated in poly-D-lysine-coated 12-well plates or 35-mm dishes. Microglia cells were allowed to attach to the plate surface for 1 h at 37°C. Following 1 h of incubation, unattached cells were washed away. Cells in 35-mm dishes were allowed to grow for 2–3 d and used for the in vitro phagocytosis assay.

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