70 mM, as this corresponds to the blood alcohol concentration of a heavy drinker, and treatments using concentrations above 100mM are known to cause a direct cytotoxic effect on the cells [38]. We chose a 24hr ethanol exposure to mimic binge alcohol drinking [34], and a 7d exposure (with daily replenishment of ethanol-containing medium) to recapitulate mild, recurring alcohol exposure. The gradual decrease in ethanol concentration in the medium due to evaporation in an unsealed culture dish follows a trend that is predicted to mimic the physiological oscillations of blood alcohol concentration in a heavy drinker with daily alcohol consumption [38–41]. Nevertheless, we acknowledge that it is possible that we didn’t achieve the intended effect of chronic, continuous alcohol exposure at higher doses. However, it is interesting to note that no difference was noticeable between acute and prolonged treatment in terms of markers for neuroinflammation, suggesting that an acute exposure to ethanol is sufficient to activate the inflammasome-mediated pathway.
