Isolation of Microglia from Mouse Brain

Microglial cells were isolated from brains as we described previously [17 (link)]. The overview of the method is depicted in Figure 1. Briefly, after perfusion with ice-cold PBS, brains were dissected, weighed, and enzymatically digested using Neural Tissue Dissociation Kit (Miltenyi Biotec, Germany) for 35 min at 37°C (if necessary, the digestion can be performed on ice, but this extends the digestion time). Further processing was performed at 4°C. Tissue debris was removed by passing the cell suspension through a 40 μm cell strainer. After myelin removal (see below), cells were stained with PE-conjugated anti-CD11b antibodies (Miltenyi Biotec, Germany) in IMAG buffer (PBS supplemented with 0.5% BSA and 2 mM EDTA) for 10 minutes followed by incubation for 15 minutes with anti-PE magnetic beads. CD11b⁺ cells were separated in a magnetic field using MS columns (Miltenyi Biotec, Germany). The amounts of antibodies and magnetic beads were calculated based on the number of cells obtained after myelin removal, using the manufacturer’s guidelines. Both the CD11b⁺ and CD11b^- (effluent) fractions were collected and used for further analyses.