FACS analysis
DNA content was analysed using the propidium iodide (PI) staining assay.
Cells were first recovered from the glycerol stocks on YPD solid media
and incubated overnight at 30 °C. The following day, a fraction of each
patch was taken with a pipette tip, transferred into liquid YPD media,
and incubated overnight at 30 °C. Then, 1 mL of each culture was taken
and resuspended in 2.3 mL cold 70% ethanol. Cells were fixed overnight
at 4 °C, washed twice with PBS, resuspended in 100 μL staining solution
(15 μM PI, 100 μg mL-1 RNase A, 0.1% v/v Triton-X, in
PBS) and finally incubated for 3 h at 37 °C in the dark. Ten thousand
cells for each sample were analysed on a FACS-Calibur flow cytometer.
Cells were excited at 488 nm and fluorescence was collected with an
FL2-A filter. Three strains with known ploidy were used as a control:
Two S. euabaynus (n and 2n) and one S. cerevisiae (4n).