Microglia were exposed to ethanol in incubator chambers (Modular Incubator Chambers MIC-101, Billups-Rothenberg Inc. Del Mar, CA) containing either 100 mL ddH2O alone or 75 mM ethanol. Ethanol was added directly to the cell media to bring the final concentration to 75 mM. The chambers were flushed with 5% CO2, 21% O2, balanced nitrogen mixture from a compressed air tank at 0.5 psi for 4 min, and then incubated at 37 °C for 24 h. Where indicated, a mixture of pro-inflammatory cytokines (“TII”, TNFα, 10 ng/ml; IL-1β, 10 ng/ml; and IFNγ, 10 IU/ml) dissolved in cell culture media was added to the cells to induce an inflammatory response; control cells received the equivalent volume of cell culture media.
