Cultures were fed daily for 7 days with neural differentiation media containing 50 mM ethanol (0.23 mg/dl). Evaporation of alcohol following daily media changes in unsealed culture plates is expected to produce a gradual reduction of alcohol each day. Evaporation of alcohol was examined in sister culture dishes not containing neural cells by measurement of media alcohol concentrations at 2, 4, 6, 8, 10 and 24 hours post-feeding using an AM1 Alcohol Analyzer (Analox Instruments Ltd, The Vale, London, UK). Alcohol concentration was gradually reduced with a ~19-hour half-life, such that by the end of 24 hours the average alcohol concentration was 18.1±2.0 mM (0.07 mg/dl). This daily replenishment of alcohol followed by gradual loss to evaporation in unsealed culture dishes provides a pattern of exposure more similar to that in human daily heavy drinkers than would continuous exposure to 50 mM ethanol in a sealed culture system.
