Name: Measuring Replicative Life Span in the Budding Yeast (Video) | JoVE Journal
Uploaded: 2009-06-25T00:00:00.000+00:00
Duration: 12 min 41 s
Description: Learn to measure replicative lifespan in budding yeast mother cells using microdissection and microscopy techniques. This protocol enables researchers to study genetic factors influencing cellular aging mechanisms.

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5 minutes

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.

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.
<ol>
	<li>Using appropriate sterile technique, prepare YEPD agar plates (1% yeast extract, 2% bacto-peptone, 2% agar, 2% glucose) that will be used for culturing yeast cells and for replicative life span analysis. You should prepare at least 2 plates for every 4-5 strains to be analyzed in the life span experiment. Plates should be prepared at least 2 days before the life span experiment and allowed to dry before beginning the life span assay. If the experiment will not be initiated within 4-5 days of pouring the plates, they should be placed in a refrigerated incubator and wrapped in parafilm or plastic to prevent dessication.</li>
	<li>Remove yeast strains from frozen stocks and streak for single colonies onto YEPD agar plates. Up to 6 strains can easily be streaked on the same YEPD plate by partitioning the plate into equally sized pie-shaped wedges.</li>
	<li>Incubate the cells at 30°C for 2 days.</li>
	<li>Remove cells from the incubator and patch cells from a single colony onto a fresh YEPD plate. Two patches from two different colonies should be generated for each strain. At this time, the strains to be analyzed should be coded to ensure the replicative life span experiment is performed "blind", where the dissectors do not know the identity of individual strains.</li>
	<li>Incubate the patched, coded cells at 30°C until the following evening.</li>
	<li>Remove the cells and lightly patch cells from each coded strain to fresh YEPD plates, which will serve as the experimental plates used for the replicative life span analysis. Cells should be patched along a vertical line on the left side of the YEPD plate (Figure 1). Approximately 3-5 patches per plate is optimal, assuming replicative life span analysis on 20 cells from each patch. It is not necessary (nor desirable) to transfer a large number of cells to the fresh YEPD plate, as this will cause the cells to grow thickly during the overnight incubation, which may limit their subsequent replicative life span.</li>
	<li>Incubate the freshly patched plates overnight on the bench top.</li>
</ol>
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.
<ol>
	<li>Using a microscope equipped with a microdissection apparatus suitable for yeast, transfer approximately 50 cells from the first patched strain to a position on the plate distal to the patched cells . If your microscope stage is graded, these gradations can be used to ensure the cells will be easy to find during subsequent iterations. On the Zeiss Axioscope 40, cells from the first patched strain can be placed at coordinates 90 x 10 and arrayed vertically from there.</li>
	<li>Clean the needle of cells by repeatedly touching the agar surface, then create a hole in the agar by forcing the needle through the agar surface. This hole will serve as a marker to orient you on the plate during subsequent iterations of dissection and daughter cell removal. Be careful not to get yeast cells in the hole, or they will grow and form a colony.</li>
	<li>Directly above the hole, vertically align individual yeast cells into 20 evenly spaced positions, with 1-3 needle diameters (~100 μM) between each cell position (Figure 1). Every few cells, place two cells adjacent to each other in the same position in the line rather than one. This allows for redundancy during virgin daughter cell selection, if one of the transferred cells fails to divide.</li>
	<li>Between the 10th and 11th positions in the line, create a horizontal series of 7-8 holes in the agar. This will serve as a marker to orient you on the plate during subsequent iterations of dissection and daughter cell removal. Be careful not to get yeast cells in the holes, or they will grow and form a colony.</li>
	<li>Repeat steps 2.1-2.4 for each patch on the plate, continuing to array cells vertically along the same axis. For step 2.2, the number of holes created should correspond to the patch number (one hole for the first patch, two holes for the second, etc.).</li>
	<li>Once cells have been arrayed for each patch, parafilm the plate and incubate at 30°C for approximately 2 hours.</li>
	<li>Repeat steps 2.1-2.6 for each plate in the experiment.</li>
</ol>
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.
<ol>
	<li>Remove the plates from the incubator and verify that most of the arrayed cells have undergone a cell division. If additional time is needed to allow cell division to be completed, return the plate to the incubator.</li>
	<li>Beginning with the first arrayed cell, use the needle to detach daughter cells from the mother cell by gently placing the needle on top of the attached cells. It is sometimes helpful to tap the side of the microscope base while the needle is resting on the cells.</li>
	<li>Place the detached daughter cell in the vertical line, replacing the mother cell and any additional daughter cells and moving them temporarily aside.</li>
	<li>Repeat step 3.2 and 3.3 for each of the arrayed cells. If a cell fails to divide, take a daughter cell from one of the redundant cells positioned in step 2.3. When finished, you should have 20 daughter cells for each patch aligned vertically on the life span plate.</li>
	<li>Collect all of the mother cells and extra daughter cells into a pile and transfer them back to an area near the patches ("the graveyard"). It is important to keep unwanted cells far from the cells undergoing life span analysis to prevent formation of microcolonies and depletion of local nutrients.</li>
	<li>Parafilm the plate and incubate at 30°C for approximately 2 hours.</li>
	<li>Repeat steps 3.2-3.6 for each plate in the experiment.</li>
</ol>
Part 4: Measuring the replicative capacity of individual cells
<ol>
	<li>Prepare the life span data sheets. A life span data sheet can be a simple grid where each row corresponds to an individual mother cell and each column corresponds to an age-point (Figure 2). Label each data sheet according to the number of strains to be analyzed.</li>
	<li>Remove the plates from the incubator and verify that most of the arrayed cells have undergone at least one cell division. If additional time is needed to allow cell division to be completed, return the plate to the incubator.</li>
	<li>Beginning with the first arrayed cell on the first plate, visually observe the mother and daughter cell(s) and determine the number of cell divisions that have occurred. It is generally easy to determine how many daughter cells a mother has produced, based on characteristic patterns of cell arrangements. Record the number of daughter cells produced by the mother cell in the appropriate box on the life span score sheet.</li>
	<li>Use the needle to detach daughter cells from the mother cell as described in step 3.2.</li>
	<li>Retain the mother cell in its position in the vertical line and move the daughter cell(s) to temporarily aside.</li>
	<li>Repeat steps 4.3-4.5 for each of the arrayed cells.</li>
	<li>Collect all of the discarded daughter cells and move them to the 'graveyard', located near the original patches.</li>
	<li>Parafilm the plate and incubate at 30°C for 2-3 hours.</li>
	<li>Repeat steps 4.3-4.8 for each plate in the experiment.</li>
	<li>Repeat steps 4.2-4.9 until all mother cells have stopped dividing. As the mother cells age, the cell cycle progression becomes slower and it will be desirable to incubate the plate for longer periods of time between age-points. Plates can be placed at 4°C overnight.</li>
</ol>
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.
<img alt="Yeast incubation process; streaking, patching, and lifespan plate diagram for colony growth study." src="/files/ftp_upload/1209/1209fig1.jpg" data-altupdatedat="1787630408000" data-alt="figure 1"> 
<img alt="Yeast cell lineage tracking, diagram shows micromanipulation setup for life span experiment." src="/files/ftp_upload/1209/fig1.2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1.5"> 
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.
<img alt="Population density diagram; grid plot shows numeric data distribution analysis, cross-hatching pattern." src="/files/ftp_upload/1209/1209fig2.jpg" data-altupdatedat="1787630408000" data-alt="figure 2"> 
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.
<img alt="Lifespan analysis graph; WT vs SIR2-OE in yeast, showing fraction viable against generations." src="/files/ftp_upload/1209/1209fig3.jpg" data-altupdatedat="1787630408000" data-alt="figure 3"> 
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.

<ol><li>Kaeberlein, M., Burtner, C. R., Kennedy, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+developments+in+yeast+aging%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">Recent developments in yeast aging.</a> PLoS Genet. 3, (2007).</li>
<li>Kaeberlein, M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKaeberlein%2C+M+%22Handbook+of+models+for+human+aging%22">Handbook of models for human aging</a>. Conn, P. M. , Elsevier Press. Boston. 109-120 (2006).</li>
<li>Smith, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+evidence+for+conserved+longevity+pathways+between+divergent+eukaryotic+species%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">Quantitative evidence for conserved longevity pathways between divergent eukaryotic species.</a> Genome Res. 18, 564-570 (2008).</li>
<li>Steinkraus, K. A., Kaeberlein, M., Kennedy, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Replicative+aging+in+yeast%3A+the+means+to+the+end%5BTitle%5D)+AND+%22Annu+Rev+Cell+Dev+Biol%22%5BJournal%5D)">Replicative aging in yeast: the means to the end.</a> Annu Rev Cell Dev Biol. 24, 29-54 (2008).</li>
<li>Murakami, C. J., Burtner, C. R., Kennedy, B. K., Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+method+for+high-throughput+quantitative+analysis+of+yeast+chronological+life+span%5BTitle%5D)+AND+%22J+Gerontol+A+Biol+Sci+Med+Sci%22%5BJournal%5D)">A method for high-throughput quantitative analysis of yeast chronological life span.</a> J Gerontol A Biol Sci Med Sci. 63, 113-121 (2008).</li>
<li>Jiang, J. C., Jaruga, E., Repnevskaya, M. V., Jazwinski, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+intervention+resembling+caloric+restriction+prolongs+life+span+and+retards+aging+in+yeast%5BTitle%5D)+AND+%22Faseb+J%22%5BJournal%5D)">An intervention resembling caloric restriction prolongs life span and retards aging in yeast.</a> Faseb J. 14, 2135-2137 (2000).</li>
<li>Kaeberlein, M., Kirkland, K. T., Fields, S., Kennedy, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sir2-independent+life+span+extension+by+calorie+restriction+in+yeast%5BTitle%5D)+AND+%22PLoS+Biol%22%5BJournal%5D)">Sir2-independent life span extension by calorie restriction in yeast.</a> PLoS Biol. 2, E296-E296 (2004).</li>
<li>Lin, S. J., Defossez, P. A., Guarente, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Requirement+of+NAD+and+SIR2+for+life-span+extension+by+calorie+restriction+in+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae.</a> Science. 289, 2126-2128 (2000).</li>
<li>Kaeberlein, M., Kennedy, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large-scale+identification+in+yeast+of+conserved+ageing+genes%5BTitle%5D)+AND+%22Mech+Ageing+Dev%22%5BJournal%5D)">Large-scale identification in yeast of conserved ageing genes.</a> Mech Ageing Dev. 126, 17-21 (2005).</li>
<li>Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+yeast+replicative+life+span+by+TOR+and+Sch9+in+response+to+nutrients%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients.</a> Science. 310, 1193-1196 (2005).</li>
</ol>

<table><tbody><tr><td>Agar</td><td>Reagent</td><td>Fisher Scientific</td><td>DF0145-17-0 (214530)</td><td></td></tr><tr><td>Bacto-Peptone</td><td>Reagent</td><td>Fisher Scientific</td><td>DF0118-17-0 (211677)</td><td></td></tr><tr><td>Yeast Extract</td><td>Reagent</td><td>Fisher Scientific</td><td>DF0886-17-0 (288620)</td><td></td></tr><tr><td>Glucose</td><td></td><td></td><td></td><td></td></tr></tbody></table>

measuring replicative life span in the budding yeast

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), 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. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

The budding yeast Croce Visi has been used extensively to study the biology of aging. In this article, we present a general protocol for measuring the replicative lifespan of yeast mother cells. Hi, I am Kristen Steffen from the laboratory of Brian Kennedy in the Department of Biochemistry at the University of Washington.Today we'll show you a procedure for measuring replicative lifespan of yeast mother cells. We use this procedure in our laboratory to study genes which influence the lifespan of yeast cells. So let's get started.For the replicative lifespan experiment and the preparation of yeast cells for lifespan analysis, solid YEPD plates need to be prepared to do this. Use appropriate sterile technique to prepare these YEPD agar plates. Prepare at least two plates for every four to five strains to be analyzed in the lifespan experiment.These plates should be prepared at least two days before the lifespan experiment and allowed to dry before beginning the lifespan assay. If the experiment will not take place within four to five days of pouring the plates, they should be wrapped in para film or plastic and be placed in a refrigerated incubator to prevent desiccation. Remove the yeast strains from frozen stocks and streak for single colonies.Onto the YEPD agar plates. Up to six strains can easily be streaked on the same YEPD plate by partitioning the plate into equally sized pie-shaped wedges. Incubate the cells at 30 degrees Celsius for two days.After incubation, remove the cells from the incubator and patch cells from a single colony onto a fresh YEPD plate. At this time, the strains to be analyzed should be coated to ensure the replicative lifespan experiment is performed blind. Where the dissectors do not know the identity of individual strains.Chains.Next, incubate the patched coated cells at 30 degrees Celsius until the following evening. Remove the cells and lightly patch cells from each coated strain to fresh YEPD plates. These will serve as the experimental plates used for the replicative lifespan.Analysis cells should be patched along a vertical line on the left side of the YEPD plate. Do not transfer too many cells onto a single fresh YEPD plate. Since this will cause thick growth of the cells during the overnight incubation and affect their replicative lifespan, approximately three to five patches per plate is optimal.Assuming replicative lifespan analysis will be performed on 20 cells from each patch, Now the lifespan experiment is all set up and all they have to do is incubate overnight on the bench top. And then tomorrow we can start the micro dissection To position the yeast cells on the plate and obtain virgin daughter cells. For replicative lifespan analysis.Use a microscope equipped with a micro dissection apparatus suitable for yeast. Transfer approximately 50 cells from the first patch strain to a position on the plate distal to the patched cells. If your microscope stage is graded, these gradations can be used to ensure the cells will be easy to find during subsequent iterations.Sometimes cells can become stuck in the needle, which can be a problem if they come out at the wrong time. To make sure the needle is clean and free of cells, touch it to the agar surface repeatedly. Then create a hole in the agar by forcing the needle through the agar surface.This hole will serve as a marker to orient you on the plate during subsequent iterations of dissection and dotter cell removal. Be careful not to get yeast cells in the hole, or they will grow and form a colony next directly above the hole. Vertically aligned individual yeast cells into 20 evenly spaced positions with one to three needle diameters between each cell position.For every few cells, place two cells next to each other in the same position in the line rather than one. This allows for redundancy during virgin daughter cell selection. If one of the transferred cells fails to divide between the 10th and 11th positions in the line, create a horizontal series of seven to eight holes in the agar.This will serve as a marker to orient you on the plate during subsequent dissections and daughter cell removals. Again, be careful not to get yeast cells in the holes or they will grow and form a colony. Repeat this procedure for each patch on the plate, continuing to array the cells vertically along the same axis.It is important to remember that when creating the hole in the AER to have the number of holes created correspond to the patch number so that there is one hole for the first patch, and two holes for the second. Once the cells have been arrayed for each patch, paraform the plate and incubate at 30 degrees Celsius for approximately two hours. Remember, from this point on, all plates should be para filmed except when undergoing dissection.In order to prevent desiccation, repeat this procedure for each plate. In the experiment. For the lifespan analysis, it is important to be sure that each cell starts as a virgin daughter cell.To do this first, remove the plates from the incubator and verify that most of the array cells have undergone a cell Division. So what we have here are cells that have been aligned previously, and you can see that after two hours in the incubator, they've begun to divide. And now we have a mother cell, the larger one attached to its body, the virgin daughter cell, and that virgin daughter cell is a cell that will be used to begin the lifespan.If additional time is needed to allow cell divisions to be completed, return the plate to the incubator. Beginning with the first array cell. Use the needle to detach daughter cells from the mother cell by gently placing the needle on top of the attached cells.It is sometimes helpful to tap the side of the microscope base while the needle is resting on the cells. Next place the detached daughter cell in the vertical line of cells replacing the mother cell and any additional daughter cells and moving them temporarily aside. Repeat these steps for each of the array cells.If a cell fails to divide, take a daughter cell from one of the redundant cells positioned next to the test cell. When finished, you should have 20 daughter cells for each patch aligned vertically on the lifespan plate. Collect all of the mother cells and extra daughter cells into a pile and transfer them back to an area near the patches called the graveyard.It is important to keep unwanted cells far from the cells undergoing lifespan analysis to prevent formation of micro colonies and depletion of local nutrients. Once the cells have been separated, para fromm the plate and incubate at 30 degrees Celsius for approximately two hours. Repeat these steps for each plate in the experiment.To measure the replicative capacity of individual cells, you'll need to prepare lifespan data sheets. A lifespan data sheet can be a simple grid where each row corresponds to an individual mother cell and each column corresponds to an age point. Be sure to label each data sheet according to the number of strains to be analyzed.To begin, first, remove the plates from the incubator and verify that most of the array cells have undergone at least one cell division. If additional time is needed to allow cell division to be completed, return the plate to the incubator. Start with the first array cell on the first plate, and visually observe the mother and daughter, cell or cells to determine the number of cell divisions that have occurred.It is generally easy to determine how many daughter cells a mother has produced based on characteristic patterns of cell arrangements. This one's really simple to see how many cells it produced because there are two there and they're the same size and they're both smaller than the mother. Record the number of daughter cells produced by the mother cell in the appropriate box on the lifespan score sheet.Next, use the needle to detach daughter cells from the mother cell as described previously by gently placing the needle on top of the attached cells and tapping the side of the microscope base while the needle is resting on the cells. Keep the mother cell in its position in the vertical line and move the daughter cell or cells temporarily aside. Repeat these steps for each of the array cells.Finally collect all of the discarded daughter cells and move them to the graveyard located near the original patches. Para film the plate and incubate at 30 degrees Celsius for two to three hours. Repeat the discarding of daughter cells and incubation.Step for each plate in the experiment. Repeat this entire procedure until all mother cells have stopped dividing. Keep in mind that as the mother cells age, the cell cycle progression becomes slower and it will be desirable to incubate the plate for longer periods of time between age points afterwards, the plates can be placed at four degrees Celsius overnight.The raw data produced by a replicative lifespan experiment is a list of numbers corresponding to daughter cells produced by each mother cell at each age point. As seen here. By summing each row of the score sheet, the replicative lifespan for each mother cell is obtained.Data from other cells from the same experimental group can be pooled to generate a survival curve and to perform statistical calculations. The curve should be roughly consistent with Gomperts Mac am kinetics, showing a sigmoidal shape with low early mortality, followed by a relatively rapid decline in survival and a flattening out of the curve at later ages. We've just shown you how to perform a yeast replicative lifespan experiment.When doing this procedure, it's important to remember that the cells will continue to divide whenever they aren't in the cold. So remember to put the plates at four degrees overnight so you don't end up with too many daughter cells to count in the morning. So that's it.Thanks for watching and good luck with your experiments.

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