Replicative life span is measured in terms of the number of mitotic divisions an individual cell completes before senescence. To measure LOH rates as mother cells divide and age, logarithmically growing MEP cells were inoculated into rich media containing estradiol and aged in liquid culture. Estradiol activates the MEP, allowing mother cells to divide and age normally while daughter cells are rendered incapable of cell division. Throughout the 95-hour aging period, samples were harvested and washed to remove estradiol, then plated to solid medium for half-sector analysis. Once the MEP is inactivated by removal of estradiol, surviving mother cells can produce viable daughter cells and form colonies. Because aged mother cells represent the only cells in the aging liquid culture capable of forming colonies (Figure 1A and [16]), no fractionation of cell populations was required to isolate aged mothers for the analysis. The vast majority of colonies at each time point grew robustly and were respiration-competent. Respiration-competent colonies were easily distinguished from those incapable of respiration, which show a severe growth defect on glucose media and thus could be excluded from our analysis.
