Metaphase spread preparations and flow cytometry analysis

To prepare metaphase spreads, cell were treated with colcemid (50 ng/ml, GIBCO BRL) for 4 h at 37°C. Cells were then collected, washed with phosphate-buffered saline and incubated with 75 mM KCl at 37°C for 15 min. After fixing in Carnoy's solution (75% methanol, 25% acetic acid), cells were dropped in 20 μl-aliquots onto microscope slides and stained them with 5% Giemsa solution. Metaphase spreads were imaged under light microscopy and chromosome numbers determined. For fluorescence-activated cell-sorting analysis, cells were collected, fixed in ice-cold methanol and incubated with RNase (50 μg/ml) and propidium iodide (PI, 100 μg/ml). DNA contents were determined using a FACSCanto II flow citometer (BD Biosciences).