In the phagocytosis assay, human iPSC-derived microglia were seeded in a 96-well plate following a 7-day intermittent ethanol exposure paradigm at varying concentrations (0 mM as the control, 20 mM, and 75 mM for phagocytosis of beads, and 0 mM and 75 mM for phagocytosis of synaptosomes). On day 7 of ethanol exposure, minocycline hydrochloride (60 μM; Sigma-Aldrich, CAS:13614-98-7) and cytochalasin D (2 μM; Thermo Fisher Scientific, catalog #PHZ1063) were added 24 hours before conducting the phagocytosis assay. To visualize the cell nuclei, Hoechst stain was diluted at a ratio of 1:1000 and added to the microglia, followed by incubation at 37°C for 10 min. Zymosan bioparticles (Thermo Fisher Scientific, catalog #P35364) or synaptosomes linked with pHrodo red dye were prepared by vortexing and then subjected to ultrasonication for 5 min. These zymosan bioparticles or synaptosomes were then added to the live microglia in the live-cell imaging solution (Thermo Fisher Scientific, catalog #A14291DJ), and the cells were incubated for 2 hours at 37°C. Live-cell images were acquired using a BZ-x800 microscope equipped with Keyence BZ-x800 software for subsequent analysis and data collection.
