Cells are grown at 25°C until mid-log phase (0.5 X 10^7 cells/ml)
in YES medium and then transferred to 37°C. If the volume is large,
it is better to harvest the cells and resuspend them in pre-warmed (37°C)
medium. The cells are then incubated at 37°C for 4.5 hrs. Cell numbers
typically double, and the cells become elongated. Then the cells are harvested
and shifted to 25°C by resuspension in medium pre-adjusted to 25°C.
Cells are then collected at the desired time intervals and analyzed as desired.
The following illustration indicates the change in position of the FACS
peak as the cells move from 1N to 2N following down-shift to 25°C:
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