Microglia from Socs3fl/fl or DKO mice were seeded into a black-walled 96-well plate at the density of 1 × 105/well. The phagocytosis assay was performed using 1) the pHrodo™ Red E. coli BioParticles™ and 2) Escherichia coli (K-12 strain) Alexa Fluor 594 BioParticles™ (both from Thermo Fisher Scientific) following the manufacturer’s instructions. Culture medium with Bioparticles alone was served as background control. The pHrodo Red E.coli BioParticles do not show any fluorescence at neutral pH, but are fluorescence brightly in acidic environments (e.g., engulfed into lysosome after phagocytosis). The fluorescence intensity was measured at 0.5 h, 1 h, 2 h and 3 h after pHrodo Red E.coli BioParticles incubation using Fluostar Omega microplate reader (BMG Labtech, Ortenberg, Germany) with 550 nm excitation wavelength and a 600 nm emission filter. Alexa Fluor 594 conjugated E.coli was washed with PBS after 0.5 h incubation, and fluorescence intensity was measured with the same wavelength. Phagocytosis was calculated by subtracting the average fluorescence intensity of the no-cell background-control wells from experimental wells. Representative images were taken using the Leica DMi8 microscope.

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