Following a 7-day maturation period in DMEM/F12 medium supplemented with N2, IL-34, and GM-CSF, human-derived microglia were subjected to a 7-day chronic intermittent exposure paradigm using ethanol 200 Proof (Decon Laboratories, catalog #2716) at concentrations of 0 mM (control), 20 mM, and 75 mM, in accordance with established protocols (89–91). Because ethanol is depleted in culture by evaporation (92), we replenished half the medium daily with the 20 or 75 mM ethanol throughout the intermittent ethanol exposure period. This meticulous daily renewal of the culture medium ensured continuous intermittent ethanol exposure, enabling the investigation of the effects of ethanol on these microglia.
