The Chronological Aging Assay

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In its most general form, the chronological aging assay requires maintaining cells in a nondividing state for a prolonged period of time, while intermittently challenging a subset of the cell population for the ability to reenter the cell cycle and successfully begin vegetative growth. Several variations of this assay have been described (Fabrizio and

Longo, 2003; MacLean et al., 2001), and many others can be imagined. The most commonly utilized variation involves growing cells into stationary phase in chemically defined media with glucose as the carbon source and maintaining the cells in culture for a period of several weeks (Fabrizio and Longo, 2003). An alternative method, in which cells are grown to stationary phase in rich media and then transferred to water, has also been described. A major difference between these two methods is the metabolic state of the cells in the quiescent state; cells aged in synthetic media maintain a high metabolic rate, whereas cells transferred to water from rich media enter a so-called hypometabolic state (Fabrizio and Longo, 2003). Survival time is greatly enhanced for cells maintained in water relative to cells maintained in synthetic medial. In general, it has been observed that mutations altering chronological life span in one of these assays have the same effect on life span as measured by the other assay variant; however, this has not been examined in a systematic manner.

In addition to the composition of the culture media, another important variant in the chronological aging assay is the condition under which cells are maintained during aging. Temperature and oxygenation are two of the most important environmental parameters that can alter the rate at which cells age chronologically. The importance of temperature is demonstrated by the observation that chronological longevity of cells maintained in water is substantially reduced as temperature is increased (MacLean et al., 2001). Survival is also reduced as oxygenation is increased. For example, cells cultured in flasks on a rotating shaker have a chronological life span that is up to 50% shorter than cells cultured in a tube on a rotating drum, while cells maintained under low aeration (no agitation) in 96-well plates have a median life span that is extended.

In the chronological aging assay, viability is determined by the fraction of cells reintroduced to favorable growth conditions that successfully reenter the cell cycle (Figure 18.1). The most common method for assaying viability involves removing an aliquot of cells from the aging culture and determining the density of colony forming units (CFUs) by plating onto YPD media. Cells aged in SC media typically reach a saturation density of 5 107 CFUs/mL by the third day in culture, which can be considered the initial viability (100%). Over time, the density of CFUs will decrease, with a rate dependent on environmental and genetic parameters, but with median survival on the order of 1-3 weeks.

In most cases, viability for each strain should be determined with time points spaced every 1-2 days. When designing a chronological aging experiment, it is important to determine whether any of the strains being examined exhibits an abnormal growth rate or stationary phase density. Extreme slow growth, in particular, should be taken into consideration when defining time points for viability measurements. Each strain should be analyzed in triplicate, at least, with replicates grown as independent cultures, rather than measuring viability from the same culture in triplicate at each time point. Stochastic variability in culture conditions and the possibility of "gasping," a phenomenon where a small fraction of aged cells escapes the G0-like state and takes over the culture population (see What Do Chronologically Aged Cells Die From?), make independent replication essential.

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