Bone marrow cells were isolated using IMDM medium, and single cell suspensions were obtained by passing the bone marrow through a 70-μm cell strainer (Falcon). Nucleated cells were counted by diluting cells tenfold in a 3% solution of acetic acid with methylene blue (Stem Cell Technologies) using a Vi-Cell XR cell viability counter (Beckman Coulter). Cells were resuspended to make up 1.5 ml of IMDM containing 30 × 106 cells and 250 μl of each suspension was mixed with 250 μl of IMDM containing 2× acetaldehyde to give final concentrations of 0, 1, 2, 4 and 8 mM acetaldehyde. The cells were incubated at 37 °C for 4 h in sealed tubes, after which two tenfold serial dilutions were made. 400 μl of cells were then added to 4 ml of MethoCult M3534 (StemCell Technologies), and the total volume of each dilution was plated in two wells of a six-well plate two wells each containing 106, 105 and 104 cells, respectively. After seven days of culture at 37 °C with 5% CO2, the colonies were counted and the relative survival was plotted. Each data point represents the average of experimental duplicates carried out on three mice of each genotype.
