Quantifying Microglial Phagocytosis of Myelin and Amyloid-β

Mice were taken down 2 days after antibody dosing, and brains were dissected after PBS perfusion and dissociated with the Adult Brain Dissociation Kit (Miltenyi Biotec, 130-107-677), according to the manufacturer’s protocol. Microglia number was measured by FACS using CountBright Absolute Counting Beads (Invitrogen, C36950) and diluted to 500 microglia per µl in DPBS + 0.5% BSA. The resulting cell suspension was mixed with pHrodo-green labeled myelin (50 µg ml⁻¹ in DPBS + 0.5% BSA) or FAM-Aβ (200 nM in DPBS + 0.5% BSA) and incubated at 37 °C for 45 minutes with gentle mixing. Cell suspensions were washed and stained with the following antibodies in FACS buffer (1% fatty acid-free BSA and 1 mM EDTA in PBS) for 25 minutes on ice: CD11b-BV421 (BioLegend, 101251) and mouse Fc blocker (anti-mouse CD16/32, BioLegend, 101320). Cells were washed with FACS buffer, resuspended in FACS buffer with propidium iodide (Miltenyi Biotec, 130-93-233), strained through a 100-μm filter and then analyzed on a flow cytometer (BD FACSAria III). FCS files were then imported and analyzed in FlowJo software (version 10).The percentage of myelin⁺ microglia (pHrodo-green⁺, CD11b⁺) and Aβ⁺ microglia (FAM⁺, CD11b⁺) in the total CD11b⁺ microglial population was calculated. See Supplementary Methods for details on myelin debris and Aβ fibril preparation and fluorescent labeling.