Name: quantifying yeast chronological life span by outgrowth of aged cells
Uploaded: 2009-05-06T00:00:00
Description: Watch this Scientific Journal Video about Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells at JoVE.com

This work was supported by NIH Grant 1R21AG031965-01A1. M. K. is an Ellison Medical Foundation New Scholar in Aging.

Part 1: Preparation of aging cultures
<ol>
	<li>Streak strains of interest from frozen stocks onto YEPD agar plates (1% yeast extract, 2% bacto-peptone, 2% agar, 2% glucose).</li>
	<li>Incubate the cells at 30&#730;C for 48 hours or until single colonies appear.</li>
	<li>Pick single colonies and inoculate into 5 mL of YEPD liquid medium (1% yeast extract, 2% bacto-peptone, 2% glucose) in test tubes.</li>
	<li>Grow cultures overnight at 30&#730;C while maintaining constant agitation using either a shaker or roller drum...

<ol>
	<li>Steinkraus, K. A., Kaeberlein, M., Kennedy, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicative+aging+in+yeast:+the+means+to+the+end.">Replicative aging in yeast: the means to the end.</a> Annu Rev Cell Dev Biol. 24, 29 (2008).</li><li>Kaeberlein, M., Conn, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of models for human aging. , 109 (2006).</li><li>Fabrizio, P., Longo, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chronological+life+span+of+Saccharomyces+cerevisiae.">The chronological life span of Saccharomyces cerevisiae.</a> Aging Cell. 2 (2), 73 (2003).</li><li>Murakami, C. J., Burtner, C. R., Kennedy, B. K., Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+high-throughput+quantitative+analysis+of+yeast+chronological+life+span.">A method for high-throughput quantitative analysis of yeast chronological life span.</a> J Gerontol A Biol Sci Med Sci. 63 (2), 113 (2008).</li><li>Piper, P. W., Harris, N. L., MacLean, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preadaptation+to+efficient+respiratory+maintenance+is+essential+both+for+maximal+longevity+and+the+retention+of+replicative+potential+in+chronologically+ageing+yeast.">Preadaptation to efficient respiratory maintenance is essential both for maximal longevity and the retention of replicative potential in chronologically ageing yeast.</a> Mech Ageing Dev. 127 (9), 733 (2006).</li><li>Fabrizio, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+is+a+mediator+of+an+altruistic+aging+program+in+Saccharomyces+cerevisiae.">Superoxide is a mediator of an altruistic aging program in Saccharomyces cerevisiae.</a> J Cell Biol. 166 (7), 1055 (2004).</li></ol>

The high-throughput chronological life span assay described here is an effective method for quantifying the aging potential of large numbers of strains with high accuracy and precision. The primary advance of this method over classical methods for determining survival by counting colony-forming units (e.g. see 3) is the use of a shaker/incubator/plate reading device such as the Bioscreen C MBR machine to obtain high-resolution growth curves at each age-point. In direct comparison with low-throughput chronolog...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Bacto Peptone</td>
<td>Reagent</td>
<td>BD</td>
<td>211677</td>
<td>&nbsp;</td>
<tr>
<td>Bacto Yeast Extract</td>
<td>Reagent</td>
<td>BD</td>
<td>288620</td>
<td>&nbsp;</td>
<tr>
<td>Difco Agar</td>
<td>Reagent</td>
<td>BD</td>
<td>214530</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Nitrogen Base w/o A.A. and A.S.</td>
<td>Reagent</td>
<td>MidSci</td>
<td>J630-500G</td>
<td>&nbsp;</td>
<tr>
<td>Amino Acids</td>
<td>Reagent</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Sulfate</td>
<td>Reagent</td>
<td>Spectrum</td>
<td>AM185</td>
<td>&nbsp;</td>
<tr>
<td>Dextrose</td>
<td>Reagent</td>
<td>Fisher</td>
<td>D16-10</td>
<td>&nbsp;</td>
<tr>
<td>Bioscreen C MBR machine</td>
<td>Tool</td>
<td>Growth Curves USA</td>
<td>5101370</td>
<td>&nbsp;</td>
<tr>
<td>Bioscreen 100-well Honeycomb plate</td>
<td>Tool</td>
<td>Growth Curves USA</td>
<td>9502550</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

quantifying yeast chronological life span by outgrowth of aged cells

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and chronological life spans are two established paradigms used to study aging in yeast. Replicative aging is defined as the number of daughter cells a single yeast mother cell produces before senescence; chronological aging is defined by the length of time cells can survive in a non-dividing, quiescence-like state 2. We have developed a high-throughput method for quantitative measurement of chronological life span. This method involves aging the cells in a defined medium under agitation and at constant temperature. At each age-point, a sub-population of cells is removed from the aging culture and inoculated into rich growth medium. A high-resolution growth curve is then obtained for this sub-population of aged cells using a Bioscreen C MBR machine. An algorithm is then applied to determine the relative proportion of viable cells in each sub-population based on the growth kinetics at each age-point. This method requires substantially less time and resources compared to other chronological lifespan assays while maintaining reproducibility and precision. The high-throughput nature of this assay should allow for large-scale genetic and chemical screens to identify novel longevity modifiers for further testing in more complex organisms.

Watch this Scientific Journal Video about Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells at JoVE.com

Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

Chronological aging in yeast refers to the loss of cell viability associated with time in stationary phase. Here we describe a high-throughput method for quantitatively determining yeast chronological life span.

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and ...

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