Primary mouse microglia culture

Primary mixed glial cell cultures were prepared from the cortices of P1 OF1 mice, as described previously (Chhor et al., 2013). After 14 days, microglia were purified and pelleted via centrifugation and resuspended in Dulbecco’s modified Eagle medium/penicillin-streptomycin/10% foetal bovine serum (DMEM/PS/10% FBS) at a concentration of 4 × 10⁵ cells/ml. One or two millilitres per well of cell suspension was plated in 12-well (for qRT-PCR) or 6-well (for ELISA) culture plates, respectively. For immunofluorescence, 250 μl of cell suspension was plated in µ-Slide 8 Well Glass Bottom chamber slides (Ibidi, BioValley). Post-plating, media was replaced after 1 h. Based on previous work from our lab (Chhor et al., 2013), the day after plating, microglia were exposed for 4 h to DMEM (control) or IL-1β at 50 ng/ml, IL-4 at 20 ng/ml, diluted in DMEM. For RT-qPCR or ELISA analysis, media were removed and plates frozen at −80°C. For immunofluorescence experiments, cells were fixed at room temperature with 4% paraformaldehyde for 20 min.