Clonogenic assay

The rationale for this assay is that if inducing Bax expression (by culturing in galactose medium) induces cell death there will be few colonies formed when spreading these dead cells on glucose plates since death is irreversible. On the other hand, if Bax simply causes cell cycle arrest (a reversible process), cells should be able to re-enter the cell cycle and to proliferate when shifted back to permissive conditions (glucose medium) where Bax expression is shut off.

1. Inoculate a single colony of yeast bearing YEp51-Bax into 5 ml liquid SD-Leu medium and culture at 30°C in an incubator-shaker (Lab-line Instruments Inc., Melrose Park, IL) set at 250 r.p.m. to a mid-exponential phase (OD600=0.4-0.6).

2. Cells are harvested by centrifugation at 1000 g for 5 min and washed three times in sterilized H20 or PBS at room temperature to remove residual glucose.

3. After the final wash, cells are resuspended in 5 ml galactose-containing SD-Leu liquid medium for continuous culturing. At different times thereafter, cell densities are determined by measuring OD600, (OD^, of 1 equals about 3 X 107 cells/ml). Cells are then diluted and about 103 cells are spread on glucose-containing SD-Leu plates.

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