The Replicative Lifespan Assay

The goal of the replicative life-span assay is to determine how many times each mother cell buds before senescence. Cell division in yeast is an asymmetric budding process resulting in the production of a larger mother cell and a smaller daughter cell. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160 x) such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle (see Recommended Resources for information on obtaining needles). Currently, there is no automated method for determining replicative life span (see Developing High-Throughput Methodologies for Life-Span Analysis).

Prior to initiating the replicative life-span experiment, it is important that all strains to be analyzed are freshly removed from frozen stock and allowed to enter a logarithmic growth phase for several divisions, as growth into stationary phase is known to depress repli-cative life span (see The Replicative Life-Span Assay). The standard growth conditions for replicative life-span experiments are growth on solid YPD (2% bacto peptone, 1% yeast extract, 2% glucose, 1.7% agar) plates at 30°C. Replicative life-span analysis can also be carried out on synthetically defined medium; however, this is generally not recommended as growth on synthetic media shortens life span dramatically (Jiang et al., 2000), complicating phenotypic analysis (Kaeberlein et al., 2004b). Cells can be placed at 4-10°C overnight, or the experiment can be carried out continuously by maintaining optimal growth temperature. It has been observed in multiple wild-type backgrounds that overnight incubation at a reduced temperature has no significant effect on longevity (Kennedy et al., 1994); although, it is possible that certain mutant backgrounds could be differentially affected by growth at reduced temperature.

There are a variety of similar methods that can be used to prepare cells for a replicative life-span experiment. One preferred method is to remove the strains from frozen stock and allow the formation of small colonies on YPD plates. Cells from a single colony are then patched lightly onto fresh YPD and cultured overnight. The following evening, cells are again patched lightly onto fresh YPD and allowed to grow for approximately 8-12 hours. The patches should be placed near one edge of the plate, and, depending on the number of cells to be analyzed per strain, up to 5 patches can be arranged on the same plate (Figure 18.4).

From this point on, it is essential to keep the plates wrapped in parafilm at all times, except during

/ Cells for

/ life span

/ assay

STRAIN #1 —14 STRAIN #2 | > STRAIN #3 —V->

\ 'THE GRAVEYARD

Figure 18.4. Yeast replicative life-span assay. Strains are lightly patched onto a 100-mm diameter YPD plate and allowed to grow overnight. 40-50 cells from each strain are arrayed for life-span analysis away from the patches. Daughter cells are discarded in ''the graveyard'' throughout the experiment.

micromanipulation, in order to prevent moisture loss and excessive drying of the agar.

Following the overnight incubation, the cells should be growing under near optimal conditions. The next step is to position the cells away from the patches for life-span analysis. For each strain, transfer approximately 100 cells to a position on the plate distal to the patched cells using the dissecting needle. Then arrange the appropriate number of cells (generally 40-50 per strain) in a line with 1-3 needle diameters (~100 ^m) between each cell. The cells should be allowed to go through at least one division (~2 hours at 30°C), at which time daughter cells should be selected and the mother cells discarded. This guarantees that every cell for which replicative life span is determined started as a virgin cell. From this point on, the life-span assay is an iterative process of allowing the cells to divide 1-2 generations in the incubator; dissecting daughter cells away from mother cells and counting the number of daughters produced by each mother; and discarding daughter cells by moving them to a distal portion of the plate (''the graveyard''). Once all of the mother cells have failed to divide after incubation for greater than 8 hours at 30°C, the experiment is complete. Statistical analysis of replicative life-span data is generally carried out using a Wilcoxon Rank-Sum test to determine whether a significant change in median life span has been observed.

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