In this article we present a general protocol for measuring the replicative life span of yeast mother cells.
Part 1: Prepare strains and plates for replicative life span analysis
This section describes the preparation of the solid YEPD plates for use in the replicative life span experiment and the preparation of yeast cells for life span analysis.
Part 2: Position cells for replicative life span analysis.
In this section we describe how to position the yeast cells on the plate and how to obtain virgin daughter cells for replicative life span analysis. From this point on, all plates should be parafilmed, except when undergoing dissection, in order to prevent dessication.
Part 3: Obtain virgin daughter cells for life span analysis
In this section we initiate the life span analysis by ensuring that each cell starts as a virgin daughter cell.
Part 4: Measuring the replicative capacity of individual cells
Part 5: Representative Results.
The raw data produced by a replicative life span experiment is a list numbers corresponding to daughter cells produced by each mother cell at each age-point (Figure 2). By summing each row of the score sheet, the replicative life span for each mother cell is obtained. Data for mother cells from the same experimental group can be pooled to generate a survival curve (Figure 3A) and to perform statistical calculations, such as mean life span, median life span, and standard deviation. A non-parametric test, such as the Wilcoxon Rank-Sum test, can be performed to determine whether significant differences in life span were detected between experimental groups. When the experiment works correctly, the survival curve should be roughly consistent with Gompertz-Makeham kinetics showing a sigmoidal shap with low early mortality followed a relatively rapid decline in survival and a flattening out of the curve a later ages. Most wild-type strains have replicative life span values in the 20-30 generation range, although variation outside of this range has been reported.
Figure 1. Diagram of a typical replicative life span plate. The evening before beginning microdissection of yeast cells, 3-5 strains are lightly patched onto the surface of a YEPD plate (red boxes) in a vertical line along one side of the plate. Following growth overnight, approximately 30 individual cells from each strain are arrayed vertically into a line containing 20 positions. Life span analysis will be performed on v virgin daughter cells obtained from these initial cells. Extra cells obtained from microdissection during the experiment (e.g. unwanted daughter cells) are placed in "the graveyard" (gray oval), which is located far away from the experimental cells in order to avoid local depletion of nutrients as colonies are formed.
Figure 2. A replicative life span tally sheet. Raw data from a replicative life span experiment is shown. Each row corresponds to a single yeast mother cell and each column corresponds to an age-point. The number entered in each box is the number of daughter cells produced by that mother cell at that age-point. Each strain is labeled with a coded number such that the individual dissecting the yeast cells has no knowledge of the identity of the strains being analyzed. Twenty cells are assayed for each strain.
Figure 3. Representative survival and growth curves from a replicative life span experiment. (A) Survival curves are obtained by plotting the fraction of mother cells still alive as a function of replicative age (number of cell divisions. (B) Growth curves are obtained by plotting the average number of daughter cells produced by a given strain as a function of age-point. In this example, strain #2 is longer-lived but is slower growing, as evidenced by the reduced initial slope of the growth plot.
This work was supported by a grant to M.K and B.K.K. from the Ellison Medical Foundation. M. K. is an Ellison Medical Foundation New Scholar in Aging. We would like to thank Soumya Kotireddy for assistance during filming.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
Agar | Reagent | Fisher Scientific (BD Diagnostic Systems) | DF0145-17-0 (214530) | |
Bacto-Peptone | Reagent | Fisher Scientific (BD Diagnostic Systems) | DF0118-17-0 (211677) | |
Yeast Extract | Reagent | Fisher Scientific (BD Diagnostic Systems) | DF0886-17-0 (288620) | |
Glucose |