Cd11b conjugated beads

Microglia were isolated from adult mouse brains as previously described [30 (link)]. In brief, mice were transcardinally perfused with 0.01 M PBS followed by cortex dissection from both hemispheres. The cortices were dissociated with papain-based enzyme mix (Miltenyi, 130–092-628) using gentleMACS™ Dissociator (Miltenyi, 130-093-235) followed by debris removal through filtering with the 70-µm cell strainer. Myelin was then removed through magnetic sorting after the incubation with myelin removal beads (Miltenyi, 130-096-731). The obtained single cell suspensions were incubated with CD11B^- conjugated beads (Miltenyi, 130-049-601) followed by magnetic sorting for CD11B⁺ microglia. Around 300 K microglia were captured per sample and lysed with RIPA buffer supplemented with the protease and phosphatase inhibitors followed by mild agitation at 4 °C for 30 min and centrifugation at 20,000 g at 4 °C for 30 min. Supernatant was collected as RIPA lysate and subject to Western blotting.

Microglia isolation was performed on 7-month-old 5xFAD and wt mice. Mice were terminally anesthetized and decapitated, and the brains were excised into Earl’s based salt solution. The brains were dissociated to a single-cell suspension using the Neural Tissue Dissociation Kit (P) (Miltenyi Biotec, 130–092–628) according to manufacturer’s protocol. For myelin removal, Percoll (GE Healthcare) protocol was performed. Microglia were isolated from myelin-free single-cell suspension using CD11b-conjugated beads (Miltenyi Biotec, 130-093-634) according to manufacturer’s protocol followed by depletion with MS columns (Miltenyi Biotec, 130–042–201). The cells were counted followed by either RNA extraction or seeded for in vitro ROS and NO measurements.