Immortalized lymphoblastoid cell lines (LCLs) were created from peripheral blood mononuclear cells isolated from subjects recruited as part of the Collaborative Study on the Genetics of Alcoholism (Begleiter et al., 1995; Bierut et al., 2010; Edenberg and Foroud, 2006). Immortalization was by transformation with Epstein-Barr virus and early passage (>12) cultures were used. In a test of the effects of ethanol on cell growth, 2 × 106 LCLs from each of three individuals were cultured in the presence of 0, 50, 75 or 100 mM ethanol in 10 ml RPMI1640 medium supplemented with 15% FBS, 2 mM glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin at 37C. For each treatment (cell line and ethanol concentration) 5 identical parallel flasks were seeded. At a given time, cells in one flask were counted twice, and the average number used to calculate a growth curve and doubling time for each individual.
