This Protocol was originally developed at Fred Hutchinson Cancer Research Center in Seattle by Xiaolan Ye and Michael Petrascheck in the Laboratory of Linda Buck. The detailed version above has been prepared to make the entire procedure available to the wider community. We thank Carol Taylor, Sarah LeBoeuf and Andy Tomacelli for critically reading the protocol. This is manuscript #2866 from The Scripps Research Institute. The Petrascheck Lab is funded by the Novartis ADI program.

Overview: The protocol is split into four parts. Part 1 describes how to prepare the feeding bacteria. Part 2 describes how to prepare the worm culture. Part 3 describes how to score lifespan. Part 4 shows some representative results, and Part 5 describes how to prepare the required solutions. Lifespan experiments take several weeks to complete. For each step of the protocol week, day, and time of the day is included to facilitate planning. The L4 stage (Day 0) is used as the reference point for the entire protocol.
1. Preparation of Feeding Bacteria
This section describes the preparation of the feeding bacteria. The specific E. coli strain used to feed C.elegans is called OP50. Prior to this protocol, to prevent cross contamination of the worm culture with other bacteria, the OP50 strain has been made Carbenicillin/Ampicillin resistant21. Prepare the OP50 four to five days in advance. All materials coming in contact with OP50 must be sterile.
Day -7: Thursday (week 1): Inoculate 5 mL of TB containing 100 &mu;g/ mL Ampicillin and 0.1 &mu;g/ mL Amphotericin B with a single OP50 colony and incubate over night at 37&deg;C in a bacterial shaker.
Day -6: Friday (week 1).
Morning 8:30. Inoculate early to allow enough time for the culture to reach saturation
<ol>
<li>Dilute the overnight culture of OP50 1:2000 in 300 mL TB containing 50 &mu;g/ mL Ampicillin. Incubate the culture in a bacterial shaker for 8-12 hours at 37&deg;C until saturation is reached. Do not allow the culture to grow longer than 14 hours.</li>
<li>Transfer the OP50 into a sterile, pre-weighted centrifugation tube. Pellet the OP50 by centrifugation for 10 min at 3500 rpm (2200 x g) in a table top centrifuge.</li>
<li>Discard the supernatant and re-suspend the OP50 pellet in sterile water and re-pellet by centrifugation. Repeat this wash twice.</li>
<li>After the second wash, carefully remove all the remaining water. No water should be left in the tube. Weigh the centrifugation tube containing the pellet and subtract the weight of the empty centrifugation tube in order to determine the weight of the pellet.</li>
<li>Thoroughly re-suspend the pellet in S-complete to a concentration of 100 mg/ mL. No clumps should be left. </li>
<li>The concentration of 100 mg/ mL OP50 should correspond to 2 x1010 bacteria/ mL. Use a photo spectrometer to determine the number of bacteria per mL if the relationship between the optical density and number of bacteria per mL is known. If necessary, adjust the concentration of the OP50 feeding solution to 2 x1010 bacteria/ mL.</li>
<li>Store the OP50solution at 4&deg;C until it is used for the worm culture.</li>
</ol>

2. Preparation of a Synchronous Worm Culture
This section describes the preparation of the worm culture. Its goal is to generate an age-synchronous population of worms. All materials coming in contact with C.elegans after the bleach treatment in step 5 must be sterile. Plates are kept at 20&deg;C unless otherwise indicated.
Day -6: Friday (week 1), 4:00 p.m.: Transfer the animals to a fresh plate
<ol>
<li>Take a 5-10 day old NGM plate on which the majority of the worm population consists of starved L1 larvae.</li>
<li>Sterilize a metal spatula by shortly heating it over a Bunsen burner. Let it cool. Use the cooled spatula to cut out agar chunks from the plate containing the starved worms. Transfer several of these chunks onto a fresh 10 cm NGM plate seeded with OP50. Incubate for approx. 65 hours at 20 &deg;C until the majority of the worm population consists of gravid adults. The time it takes for the starved L1 animals to grow into gravid adults may vary from strain to strain.</li>
</ol>
Day -3: Monday (week 2) 10:00 a.m.: Establish a synchronous population
<ol start="3">
<li>Collect worms from the 10 cm plate by washing them off the plate with 5-10 mL sterile water. Transfer the worm/water solution into a 15 mL conical tube.</li>
<li>Wash the worms by centrifuging 2 min in a tabletop centrifuge at 1200 rpm (280 x g). Discard supernatant and add 10 mL water. Repeat.</li>
<li>Remove supernatant and add 5 mL of freshly prepared bleach/NaOH solution (2.5 mL household bleach, 1 mL 10N NaOH, 6.5 mL H2O). Incubate for 5 minutes at RT until the worms break open. Be sure to vortex gently every minute. Monitor the progress of the reaction under the dissecting microscope.</li>
<li>As soon as all the adults dissolve, add 5 mL of M9 buffer to neutralize the reaction.</li>
<li>Wash the eggs three times with 10 mL M9 buffer by centrifuging 2 min at 2500 rpm (1100 x g).</li>
<li>Wash the eggs once with 10 mL S-complete by centrifuging 2 min at 2500 rpm (1100 x g).</li>
<li>Remove the supernatant, add 10 mL S-complete and transfer the solution to a fresh 50 mL conical tube. Add 30 mL of S-complete to a final volume of 40 mL. Gently shake the tube overnight at room temperature on a nutator or similar device.</li>
</ol>
Day -2: Tuesday (week 2), 12:00 noon: Seed the animals into plates
<ol start="10">
<li>Under the dissecting scope, check whether the worms hatched during the night. Determine the concentration of worms in the S-complete solution by counting the number of worms in 10 &mu;L drops using a dissecting scope. Count at least 10 drops for each sample.</li>
<li>Re-suspend the worms at a concentration of 80-100 worms/ mL in S-complete. Add Carbenicillin (stock 100 mg/ mL) to a final concentration of 50 &mu;g/ mL and Amphotericin B (stock 250 &mu;g/ mL) to a final concentration of 0.1 &mu;g/ mL. Shake on a nutator. If preparing larger quantities of worms, use a 600 mL nunclon flask with filter cap.</li>
<li>At 2:30 p.m. add the OP50 prepared in Part 1 to a final concentration of 6 mg/ mL (= 1.2 x109 bacteria/ mL). Return the OP50 to 4&deg;C.</li>
<li>Transfer 120 &mu;L of the worm/OP50 solution into each well of a 96 well plate. Use 96 well plates with a transparent bottom. Make sure to keep the worms in suspension while pipetting.</li>
<li>Seal the plate using a tape sealer to avoid contamination and evaporation. Shake the plate on a microtiter plate shaker for 2 min and incubate for 2 days at 20&deg;C until the animals reach the L4 stage.</li>
</ol>
Day 0: Thursday (week 2) before noon: Sterilize animals by adding Fluorodeoxyuridine (FUDR)
<ol start="15">
<li>To sterilize the animals add 30 &mu;L of a 0.6 mM FUDR stock solution to each well. This step brings the final volume in each well to 150 &mu;L and reduces the final concentration of OP50 from 6mg/ mL to 5 mg/ mL (1x109 bacteria/ mL). Seal the plate using tape sealers and shake it for 2-3 min on a microtiter plate shaker. If the OP50 was added at 2:30 on day -2, it is important that the FUDR is added before noon. Return plates to the 20&deg;C incubator</li>
</ol>
Day 1: Friday (week 2): Add drugs to culture
<ol start="16">
<li>By 9:00 a.m. most of the animals should be gravid and contain several eggs each. Add the drugs whose effect on lifespan is to be tested at the desired concentration. If dissolving the drugs in DMSO the final concentrations of DMSO should not exceed 0.6%, as DMSO concentrations higher than 0.6% shorten C.elegans lifespan. After addition of the drug, seal the plates with tape sealer and shake it for 2-3 min on a microtiter plate shaker. Return plates to the 20&deg;C incubator</li>
<li>Adding the drugs can occasionally kill a few animals per plate, especially if using a solvent other than water. Use an inverted microscope to check for dead animals. Generally, there should be less than 10 dead animals per 96 well plate. Return plates to the 20&deg;C incubator.</li>
</ol>
Day 4: Monday (week 3): Change sealers
<ol start="18">
<li>To allow fresh oxygen to enter the culture remove the sealer, wait 1minute and reseal the plate. Shake the plate for 2-3 minutes on a microtiter plate shaker. Repeat once every week.</li>
</ol>
Day 5: Tuesday (week 3): Add new OP50 to prevent starvation
<ol start="19">
<li>Add 5 &mu;L of the 100 mg/ mL OP50 solution that was prepared in part 1 to each of the 96 wells to prevent starvation.</li>
</ol>
3. Scoring of Lifespan.
This section describes how survival of the synchronized worm population of Part 2 is monitored until the animals died. To observe the animals in the 96 well plates, use an inverted microscope with a 2x or 2.5x objective. Record survival data three times a week; Monday, Wednesday and Friday. Use movement to determine whether the animals are alive or dead. Strong light, especially blue light, induces the animals to move. Do not remove dead animals from the assay. Occasionally, animals that did not move and were determined dead in the preceding count might move later on.
<ol>
<li>On day 0, at the beginning of the experiment count the total number of worms in each well. Censor wells that contain more than 18 animals from the analysis as animals in these wells will not have enough OP50 and will show effects of dietary restriction.</li>
<li>In order to increase the chance that the animals move shake the 96 well plate on a microtiter plate shaker for 2 min. before counting.</li>
<li>In each counting session, record date and number of animals which move as live animals. Movement in liquid is much easier than on solid media and can be induced by strong lights. The use of a higher magnification may help to spot very subtle movements like those of the tip of the pharynx. Such subtle movements are often the only movement observed in very old animals.</li>
<li>Return plates to the 20&deg;C incubator</li>
<li>Repeat step 2-4 every two to three days until all the animals are dead.</li>
</ol>
4. Representative Results.

This section shows an example of how to keep records of the lifespan data generated by this assay and some representative results.
Figure 1A shows an example of how to record lifespan data during this assay. An excel sheet is used to keep track of the survival the populations in each well. For each well it's coordinate in the plate, strain, drug, concentration of the drug and the total number of animals alive on day 0 (X0) is recorded at the beginning of the experiment. Record date, as well as the number of living and dead animals three times a week in order to follow the survival of the various populations in each well. To graph the results, calculate the fraction of animals alive for each day and plot it as a function of time in days. P-values should be calculated using statistical packages like STATA or similar software.
The medium lifespan of C.elegans is temperature dependent. Figure 1B shows that temperature dependent changes in lifespan are accurately reproduced by the microtiter based lifespan assay22. Similarly, the microtiter plate assay reproduces changes in lifespan from mutants reported to have lifespans that differ from wild type N2 animals23-24 25(Fig 1C).
The assay also can be used to make more quantitative statements. In Fig1D mean lifespan is plotted as a function of Mirtazepine concentration7. Each data point represents the mean lifespan of 7-12 population, each living in a different well. Even though the number of animals per well is relatively low (5-15 animals) the well-to-well variation is relatively small as can be seen from the error bars.
 
 <img src="/files/ftp_upload/2496/2496fig1.jpg" alt="Figure 1" /> 
Figure 1. The microtiter plate based lifespan assay accurately reproduces changes in lifespan reported in the literature. 
(A) Sample data collected in an excel spreadsheet. During each counting session date, coordinate of the well and the number of live and dead animal was recorded. At the beginning of the experiment, the total number of animals in each well was determined. (B) Survival curve of wild type (N2) animals grown at 20&deg;C and 25&deg;C. (C) Survival curves of strains carrying mutations that affect lifespan. All four strains were assayed in parallel at 20&deg;C. (D) Dose response curve of Mirtazepine treated N2 animals. Mean lifespan is plotted as a function of Mirtazepine concentration. Error bars indicate the S.E.M of 8 wells per condition. 

 
5. Materials.


M9 buffer, 1000 mL
<ul>
<li>6 g Na2HPO4</li>
<li>3 g KH2PO4</li>
<li>5 g NaCl</li>
<li>0.25 g MgSO4&#8729;7H2O</li>
<li>Add deionized water to 1000 mL </li>
<li>Autoclave</li>
</ul>
Potassiumphosphate buffer pH 6.0, 1000 mL
<ul>
<li>136 g KH2PO4</li>
<li>Add deionized water to 900 mL</li>
<li>Adjust pH to 6.0 with 5M KOH</li>
<li>Add deionized water to 1000 mL</li>
<li>Autoclave</li>
</ul>
Trace metal solution
<ul>
<li>1.86 g Na2EDTA</li>
<li>0.69 g FeSO4 &#8729;7H2O</li>
<li>0.20 g MnCl2 &#8729; 4H2O</li>
<li>0.29 g ZnSO4 &#8729;7H2O</li>
<li>0.016 g CuSO4</li>
<li>1000 mL deionized water</li>
<li>Autoclave and store in the dark.</li>
</ul>
S-basal medium, 1000 mL
<ul>
<li>5.9 g NaCl</li>
<li>50 mL of 1M potassium phosphate, pH 6.0</li>
<li>1000 mL deionized water</li>
<li>Autoclave</li>
<li>Let the solution cool and then add 1 mL of 5mg/ mL cholesterol (dissolved in Et-OH).</li>
</ul>
Potassium citrate 1M, 1000 mL
<ul>
<li>268.8 g tripotassium citrate</li>
<li>26.3 citric acid monohydrate</li>
<li>Add 900 mL deionized water</li>
<li>Adjust pH to 6.0 with 5M KOH</li>
<li>Add deionized water to 1000 mL</li>
<li>Autoclave</li>
</ul>
S-complete medium, 1000 mL
<ul>
<li>977 mL S-basal</li>
<li>10 mL 1M potassium citrate pH 6 (sterile)</li>
<li>10 mL Trace metals solution (sterile)</li>
<li>3 ml 1M CaCl2 (sterile)</li>
<li>3 mL 1M MgSO4 (sterile)</li>
</ul>
NGM agar
<ul>
<li>3.0g NaCl</li>
<li>2.5g Pepton (from casein, pancreatic digest)</li>
<li>17g Agar</li>
<li>Add deionized water to 975 mL and a stirring bar</li>
<li>Autoclave</li>
<li>After autoclaving cool down to 55&deg;C and add the following components:
<ul>
<li>0.5 mL of 1M CaCl2 (sterile)</li>
<li>1 mL of 5mg/ mL Cholesterol in ethanol</li>
<li>1 mL of 1M MgSO4 (sterile)</li>
<li>25 mL Potassiumphosphate buffer, pH 6.0 (sterile)</li>
</ul></li>
</ul>
TB, 1000 mL
<ul>
<li>12g Bacto Tryptone</li>
<li>24 g Yeast Extract</li>
<li>4 mL Glycerol</li>
<li>Add 900 mL deionized water</li>
<li>Autoclave</li>
<li>After autoclaving cool down to 55&deg;C and add the following component:
<ul>
<li>add 100 mL of 0.17M KH2PO4/0.72M K2HPO4</li>
</ul></li>
</ul>
0.6 mM Fluorodeoxyuridine (FUDR, sigma cat# F0503), 1000 mL
<ul>
<li>100 mg FUDR</li>
<li>Dissolve in 670 mL sterile S-complete, make 10 mL or 45 mL aliquots.</li>
<li>Store at -20 &deg;C.</li>
</ul>
100 mg/ mL Carbenicillin, 10 mL
<ul>
<li>1 g Carbenicillin</li>
<li>10 mL sterile deionized water</li>
<li>Sterile filtrate and aliquot</li>
<li>Store at -20 &deg;C.</li>
<li>Use at a final concentration of 50 &mu;g/ mL</li>
</ul>
250ug/ mL Amphotericin B, 4 mL
<ul>
<li>1mg Amphotericin B</li>
<li>4 mL Et-OH</li>
<li>Store at -20 &deg;C</li>
<li>Use at a final concentration of 0.1 &mu;g / mL</li>
</ul>

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No conflicts of interest declared.

The protocol presented allows the measurement of C.elegans lifespan in 96 well microtiter plates. As shown in the representative results section it reliably replicates previous findings and provides quantitative information. Using this assay we have successfully screened over 89,000 molecules for their effect on C.elegans lifespan.
For the purpose of drug-screening, measuring lifespan in a 96 well microtiter plate format has several advantages over the classical solid media assay. It reduces the labor required for media preparation, the amount of incubation space, and the amount of drug required. The 96-well format and the microscopy setup allow automation of the entire assay for high throughput screenings.
During the development of the assay multiple values for each variable and combinations thereof were tested for their effects on C.elegans lifespan. These tests included different OP50 concentration ranging from 3 to 10 mg/ mL (3, 4, 6, 8, 10 mg/ mL), different number of worms per well ranging from 7 to 45 (7, 10, 15, 22, 45 worms/well), different culture volumes ranging from 40 to 150 &mu;L per well (40, 60, 80, 100, 120, 150 &mu;L), and changes in buffer composition. If fed with a Carbenicillin-resistant OP50, neither Carbenicillin nor Amphotericin B in the concentrations indicated were found to affect C.elegans lifespan. Continuous gentle shaking of the plates, as is often recommended in C.elegans liquid culture, was found dispensable for the small volumes used in this assay. Gentle shaking however is required if microtiter plates with larger wells are to be used. The values indicated in this protocol have been carefully selected based on statistical comparison of the different conditions tested.
The most surprising feature of this assay is probably the fact that C.elegans can be kept in 96 well plates that are sealed with tape. Side-by-side comparisons did not reveal any difference in lifespan of animals cultured in unsealed plates, in plates sealed with a plastic sealer, or in plates sealed with sealers that allow air exchange. In all three conditions, the animals developed very homogenously from L1 to gravid adults within 65 hours and showed very comparable lifespans. Animals grown in parallel on NGM developed slightly faster, but this difference was independent of the absence or presence of a sealer.
The presented protocol is based on live bacteria, but can be adapted for dead bacteria. However, in a liquid assay a single surviving bacteria can quickly multiply and re-populate the culture. In our hands, the only reliable way to kill bacteria to the extent that they can be used for liquid culture is by prolonged treatment of the bacteria with gamma irradiation.
One important point to consider in the planning of a lifespan experiment is the power of detection. Dependent on the size of the effect, the effect of the drug of interest must be tested in multiple wells. In a typical assay 4 drug-treated and 4 control wells, corresponding roughly to 40-50 animals each, should suffice to detect a 30% increase in lifespan in more than 95% of the experiments. Increases of 14 % are only detected in 60% of the cases and therefore require more replicate wells.
The wealth of lifespan data generated by this assay may be used to develop experiment or strain specific parametrical Gompertz models1. These Gompertz models are useful to determine the power of detection and to estimate the number of false positives and negatives for large-scale screens. We verified the predictions of these models in blind experiments and used them to estimate the numbers of false negatives in large screens (unpublished results).
In summary, we anticipate that the presented assay will be of great use to identify small molecules that extend lifespan of C.elegans and to study the underlying mechanisms.

A correction to the article Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates was made.

Erratum: Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

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<td>Amphotericin B</td>
<td>&nbsp;</td>
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<td>FUDR</td>
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<td>FUDR is inactivated by heat, thaw in cold water</td>
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<td>Mianserin</td>
<td>&nbsp;</td>
<td>Sigma-aldrich</td>
<td>M2525-250MG</td>
<td>Use as positive control at 50&mu;M final concentration</td>
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<td>Cyproheptadine</td>
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<td>Sealing Tape</td>
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<td>96 well plate</td>
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<td>Nunclon flask</td>
<td>&nbsp;</td>
<td>Nunc</td>
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measuring caenorhabditis elegans life span in 96 well microtiter plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Watch this Scientific Journal Video about Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates at JoVE.com

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

measuring-caenorhabditis-elegans-life-span-96-well-microtiter

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36.2K vues.  The Scripps Research Institute. Dans ce protocole, nous présentons une méthode pour mesurer Caenorhabditis elegans Durée de vie de 96 plaques de microtitration bien.

Vidéo: Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

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

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.

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 ...

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 ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...