This work was supported by a Glenn/AFAR Breakthroughs in Gerontology Award and NIH Grant 1R01AG031108-01 to M. K. G. S. is supported by NIH training grant P30AG013280. M. K. is an Ellison Medical Foundation New Scholar in Aging.

Part 1: Prepare nematode growth media (NGM) plates
This section describes the preparation of the solid NGM plates for use in the life span experiment. A basic life span experiment requires two types of plates: standard NGM plates, which contain no additives, and Amp/FUDR plates, which have both ampicillin (Amp) and fluorodeoxyuridine (FUDR) added to the NGM. Ampicillin is used to prevent foreign bacterial contamination. FUDR inhibits cell division, reduces egg production, and prevents eggs from hatching. The use of FUDR for longevity analysis does not affect adult life span and removes the need to transfer worms every few days in order to separate them from growing larva. Both types of plates are seeded with E. coli OP50 bacteria, which is subsequently killed by exposure to UV.
<ol>
<li>Prepare NGM (see Section 5 for recipe and storage notes) and label petri plates. You will need 1 60 mm petri plate per 10 mL of NGM. If you are starting with previously prepared solid NGM, continue to step 1.2. If you are starting with freshly autoclaved NGM, skip to step 1.4.</li>
<li>Completely melt solid NGM by microwaving on high in 30 second pulses. Swirl media between pulses to prevent pressurized gas bubbles from building up.</li>
<li>Place liquid NGM in 55 &deg;C water bath or on the bench to cool.</li>
<li>Once NGM reaches 55 &deg;C add 33 &micro;L of 150 mM FUDR and 100 &micro;L of 100 mg/mL Ampicillin per 100 mL NGM (for Amp/FUDR plates) and swirl to mix. For NGM plates without additional drugs proceed directly to step 1.5.</li>
<li>Using sterile technique, pipette 10 mL NGM into each 60 mm petri plate. Avoid forming bubbles if possible. If bubbles do form, they can be popped using a pipette tip.</li>
<li>Leave plates on the bench with lids on to allow NGM to solidify and dry. If possible, leave plates on bench for 2 days before adding bacteria, but 1 day will work if plates are needed sooner.</li>
<li>On the day before the plates finish drying, seed liquid LB culture with a single colony from a fresh streak of E. coli OP50 bacteria on LB agar. You should prepare at least 1 mL of an overnight OP50 culture per 60 mm plate.</li>
<li>Place OP50 culture in 37 &deg;C shaker and allow bacteria to grow overnight (culture should reach saturation).</li>
<li>Pellet OP50 bacteria by spinning at 3,500 g for 10 minutes.</li>
<li>Remove 90% of supernatant and resuspend bacteria to concentrate culture 10x.</li>
<li>Pipette 100 &micro;L of 10x concentrated OP50 culture into the center of solidified NGM plates. Swirl plates gently to spread out bacteria culture if needed (ideally bacteria should cover the central area of the NGM without coming near the plate walls). Try to avoid touching the pipette tip to the surface of the NGM, as flaws in the surface of the media allow the worms to burrow into the NGM.</li>
<li>Leave plates on the bench overnight to allow bacteria culture to dry on the solid NGM (typically takes about 24 hours).</li>
<li>Once dry, expose the surface of the plates to a UV dose sufficient to arrest growth of the bacteria. If you are using a Stratagene UV Stratalinker 2400: 
<ul>
<li>Place plates in the Stratalinker and remove lids</li>
<li>Close the door and turn on the Stratalinker</li>
<li>Press &lsquo;Energy&rsquo;</li>
<li>Enter &lsquo;9999&rsquo; using the keypad</li>
<li>Press &lsquo;Start&rsquo;</li>
<li>The plates will be exposed to UV for approximately 5 minutes</li>
</ul>
</li>
<li>Plates with UV-killed bacteria can be stored upside down at 4 &deg;C for up to 1 month.</li>
</ol>
Part 2: Perform a timed egg laying to acquire an age-synchronized population of animals
In this section we generate a population of worms with a common hatch-date. This is accomplished by allowing reproductively active adults to lay eggs on a plate for a defined period of time and allowing those eggs to develop.
<ol>
<li>(Optional) Transfer approximately 20 young adult worms to a fresh NGM plate without FUDR. Leave plates at 25 &deg;C in order to allow worms to propagate and eat through all of the bacterial food. After the bacterial food has been consumed newly hatched worms will enter the growth arrested dauer stage. You will start to see dauer larva around within a week, depending on how many worms you transfer to the plate initially. Dauer larva can be used for the next steps for up to 1 month.</li>
<li>(Optional) Transfer 20 to 30 dauer larva to a fresh NGM plate without FUDR. In the presence of food dauer larva will become reproductively active adults within 2 days and remain reproductively active for a few days thereafter at 25 &deg;C.</li>
<li>Transfer 10 to 15 reproductively active adults to a fresh NGM plate without FUDR. This plate is referred to as the timed egg laying (TEL) plate.</li>
<li>Leave the TEL plate at 25 &deg;C for 6 hours in order to allow the worms time to lay eggs.</li>
<li>Remove adult worms from TEL plate. Plate can be visually inspected for eggs before removing adults. If the worms have not laid a sufficient number of eggs the TEL plate can be left at 25 &deg;C for up to a total of 24 hours before removing adult worms.</li>
<li>Place TEL plate at 20 &deg;C until the eggs have hatched and the worms have developed to the L4 larval stage (this usually takes 2 days for wild type C. elegans, but can take longer for strains with slow development phenotypes).</li>
</ol>
Part 3: Score animals for life span
In this section we follow the age-synchronized population of worms from Part 2 until they die. Worms are maintained on Amp/FUDR plates to prevent egg production and bacterial contamination and are considered dead when they fail to respond to external stimuli.
<ol>
<li>Transfer L4 larva to seeded Amp/FUDR plates. For each strain or condition being tested, it is typical to set up 2-3 plates with 25-30 worms per plate. Images of the C. elegans life stages are provided for reference (Figure 1).1</li>
<li>Place Amp/FUDR plates at 20 &deg;C for 24 hours.</li>
<li>After 24 hours, visually assess worms, media, and bacteria. Transfer worms to fresh Amp/FUDR plates if any of the following is observed: 
<ul>
<li>Worms have eaten most of the bacterial food. Early in the life span experiment worms will have to be transferred 1 to 2 times a week to prevent the bacteria from being depleted.</li>
<li>A significant number of larvae are present. This is usually an indication that the worms were transferred to the Amp/FUDR plates as young adults instead of L4s and were able to lay some eggs before the FUDR took effect. The FUDR will generally prevent the larvae from growing into full adults, but occasionally a few will grow into adults and become confused with the experimental animals.</li>
<li>Live /growing bacteria are observed. Generally, the combination of Amp and UV-killing will ensure that no live bacteria contaminate the experiment, but occasionally it occurs.</li>
<li>Fungal growth is observed on the media. If caught early enough, fungal growth can usually be cut out using a pipette tip or spatula. Once it grows to be larger than a few millimeters in diameter it is usually easier to transfer the worms to a new plate.</li>
<li>Using the dissection scope, determine whether each worm is alive or dead.</li>
<li>Gently tap the plate. The worm is alive if it moves in response to the tapping.</li>
<li>If the worm does not respond to tapping the plate, zoom in on the head region.</li>
<li>Gently tap the worm&rsquo;s head with platinum transfer pick. Score the worm as dead if it does not respond by moving its head.</li>
<li>Dead worms can be removed from the plate.</li>
</ul>
</li>
<li>Record the date and the number of worms that are alive and dead.</li>
<li>Return plates to 20 &deg;C.</li>
<li>Repeat steps 3.3 through 3.6 every 2 to 3 days until all worms have died. </li>
</ol>
Part 4: Representative Results.
The raw data produced by a nematode life span experiment is a list of dates with corresponding numbers of worms that are alive and dead for each strain tested. The number of worms that die on each day is typically inverted to calculate the proportion of worms alive on each day (Figure 2A), which is plotted graphically as a survival curve (Figure 2B; the day of the timed egg laying is considered day 0). The life span for each individual worm in the study can be calculated from the count of worms that die on each day and used to calculate mean and median life span for comparison between strains. The count of number of worms alive on each day is not used directly in life span analysis. Worms maintained on solid media will occasionally &lsquo;flee&rsquo;, or crawl either up the wall of the plate or down beneath the media. The number of worms alive on each day can be used to determine how many worms fled throughout the course of the experiment. Worms that flee are typically removed from analysis. As a benchmark, the typical median life span for N2, the C. elegans wild-type strain, maintained on UV-killed bacteria at 20 &deg;C is approximately 25 days as measured from egg.
Part 5: Solutions
<table border="0">
<tbody>
<tr>
<td colspan="2">Nematode Growth Media (NGM), 100 mL:</td>
</tr>
<tr>
<td colspan="2">Combine:</td>
</tr>
<tr>
<td>0.3 g</td>
<td>NaCl</td>
</tr>
<tr>
<td>0.25 g</td>
<td>Peptone</td>
</tr>
<tr>
<td>2 g</td>
<td>Agar</td>
</tr>
<tr>
<td colspan="2">
Autoclave for 40 minutes and let cool to 55 &deg;C, then add:
</td>
</tr>
<tr>
<td>100 &micro;L</td>
<td>1 M MgSO4</td>
</tr>
<tr>
<td>100 &micro;L</td>
<td>5 mg/mL Cholesterol</td>
</tr>
<tr>
<td>100 &micro;L</td>
<td>1 M CaCl2</td>
</tr>
<tr>
<td>1.625 mL</td>
<td>1.5 M KPi pH 6.0</td>
</tr>
<tr>
<td colspan="2">
Liquid NGM can be used immediately to pour plates or allowed to solidify and stored long-term at room temperature
</td>
</tr>
</tbody>
</table>
<table border="0">
<tbody>
<tr>
<td colspan="2">Luria Broth (LB), 1L:</td>
</tr>
<tr>
<td>10 g</td>
<td>BactoTryptone</td>
</tr>
<tr>
<td>5 g</td>
<td>Yeast Extract</td>
</tr>
<tr>
<td>10 g</td>
<td>NaCl</td>
</tr>
<tr>
<td>10 mL</td>
<td>1 M Tris pH 8.0</td>
</tr>
<tr>
<td>1 L</td>
<td>deionized water</td>
</tr>
<tr>
<td colspan="2">
Autoclave and store at room temperature.
</td>
</tr>
</tbody>
</table>
<table border="0">
<tbody>
<tr>
<td colspan="2">1 M MgSO4, 300 mL:</td>
</tr>
<tr>
<td>73.95 g</td>
<td>MgSO4</td>
</tr>
<tr>
<td>300 mL</td>
<td>deionized water</td>
</tr>
<tr>
<td colspan="2">Autoclave and store at room temperature.
</td>
</tr>
</tbody>
</table>
<table border="0">
<tbody>
<tr>
<td colspan="2">5 mg/mL Cholesterol, 200 mL:</td>
</tr>
<tr>
<td>1 g</td>
<td>cholesterol</td>
</tr>
<tr>
<td>200 mL</td>
<td>100% ethanol</td>
</tr>
<tr>
<td colspan="2">
Filter sterilize and store at room temperature.
</td>
</tr>
</tbody>
</table>
<table border="0">
<tbody>
<tr>
<td colspan="2">1 M CaCl2, 500 mL:</td>
</tr>
<tr>
<td>27.75 g</td>
<td>CaCl2</td>
</tr>
<tr>
<td>500 mL</td>
<td>deionized water</td>
</tr>
<tr>
<td colspan="2">
Filter sterilize and store at room temperature.
</td>
</tr>
</tbody>
</table>
<table border="0">
<tbody>
<tr>
<td colspan="2">1.5 M KPi pH 6.0, 1L:</td>
</tr>
<tr>
<td colspan="2">Combine:</td>
</tr>
<tr>
<td>31.4 g</td>
<td>KPi dibasic</td>
</tr>
<tr>
<td>179.6 g</td>
<td>KPi monobasic</td>
</tr>
<tr>
<td>850 mL</td>
<td>deionized water</td>
</tr>
<tr>
<td colspan="2">
Heat while mixing to allow KPi to dissolve into solution. Adjust pH to 6.0 with 10 N NaOH.
</td>
</tr>
<tr>
<td colspan="2">Add deionized water to 1 L.</td>
</tr>
<tr>
<td colspan="2">
Autoclave and store at room temperature.
</td>
</tr>
</tbody>
</table>
<table border="0">
<tbody>
<tr>
<td colspan="2">
1 M Tris, pH 8.0:
</td>
</tr>
<tr>
<td>60.57 g</td>
<td>Tris</td>
</tr>
<tr>
<td>400 mL</td>
<td>deionized water</td>
</tr>
<tr>
<td colspan="2">Adjust pH to 8.0 with HCl.</td>
</tr>
<tr>
<td colspan="2">
Add deionized water to 500 mL.
</td>
</tr>
<tr>
<td colspan="2">
Filter sterilize and store at room temperature.
</td>
</tr>
</tbody>
</table>
<table border="0">
<tbody>
<tr>
<td colspan="2">
150 mM Fluorodeoxyuridine (FUDR), 10 mL:
</td>
</tr>
<tr>
<td>0.3693 g</td>
<td>FUDR</td>
</tr>
<tr>
<td>10 mL</td>
<td>sterile deionized water</td>
</tr>
<tr>
<td colspan="2">Store at -20 &deg;C.</td>
</tr>
</tbody>
</table>
<table border="0">
<tbody>
<tr>
<td colspan="2">
100 mg/mL Ampicillin (Amp), 10 mL:
</td>
</tr>
<tr>
<td>1 g</td>
<td>Ampicillin</td>
</tr>
<tr>
<td>10 mL</td>
<td>sterile deionized water</td>
</tr>
<tr>
<td colspan="2">Store at -20 &deg;C.</td>
</tr>
</tbody>
</table>
<table border="0">
<tbody>
<tr>
<td colspan="2">
50 mg/mL Carbenicillin (Carb), 10 mL:
</td>
</tr>
<tr>
<td>500 mg</td>
<td>Carbenicillin</td>
</tr>
<tr>
<td>10 mL</td>
<td>sterile deionized water</td>
</tr>
<tr>
<td colspan="2">Store at -20 &deg;C.</td>
</tr>
</tbody>
</table>
<table border="0">
<tbody>
<tr>
<td colspan="2">
1 M Isopropyl &beta;-d-Thiogalactopyranoside (IPTG), 10 mL:
</td>
</tr>
<tr>
<td>2.38 g</td>
<td>IPTG</td>
</tr>
<tr>
<td>10 mL</td>
<td>sterile deionized water</td>
</tr>
<tr>
<td colspan="2">Store at -20 &deg;C.</td>
</tr>
</tbody>
</table>
<img src="/files/ftp_upload/1152/Figure%201.png" alt="figure 1" /> Figure 1. Bright field images of C. elegans life stages, including egg, the four larval stages (L1 &ndash; L4), and adult. All panels show hermaphrodites except the lower-right, which shows an adult male (Image from Wood (1988)).
<img src="/files/ftp_upload/1152/Figure%202.png" alt="figure 2" /> Figure 2. Representative results from a C. elegans life span experiment comparing wild type strain N2 to a strain containing a mutation in the daf-2 gene. (A) A table showing collected data, including number of days since worms were eggs, number of dead worms observed on each day, and the percentage of the original sample remaining alive on each day (as calculated from the daily counts of dead worms). (B) Survival curves corresponding to the life span data provided in (A).
<img src="/files/ftp_upload/1152/Figure 3.jpg" alt="figure 3" /> Figure 3. Representative comparison of lipofuscin between young and old adult C. elegans. Bright field images are shown on left and DAPI channel fluorescence images on the right. Top panels show a 4 day old worm and bottom panels show an 11 day old worm (as measured from egg).

<ol>
	<li>Wood, W. B., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Nematode Caenorhabditis elegans. , 1-16 (1988).</li><li>Sutphin, G. L., 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=Dietary+restriction+by+bacterial+deprivation+increases+life+span+in+wild-derived+nematodes.">Dietary restriction by bacterial deprivation increases life span in wild-derived nematodes.</a> Exp Gerontol. 43, 130-135 (2008).</li><li>Garigan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+tissue+aging+in+Caenorhabditis+elegans:+a+role+for+heat-shock+factor+and+bacterial+proliferation.">Genetic analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation.</a> Genetics. 161, 1101-1112 (2002).</li><li>Chen, D., Pan, K. Z., Palter, J. E., Kapahi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longevity+determined+by+developmental+arrest+genes+in+Caenorhabditis+elegans.">Longevity determined by developmental arrest genes in Caenorhabditis elegans.</a> Aging Cell. 6, 525-523 (2007).</li><li>Curran, S. P., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifespan+regulation+by+evolutionarily+conserved+genes+essential+for+viability.">Lifespan regulation by evolutionarily conserved genes essential for viability.</a> PLoS Genet. 3, e56-e56 (2007).</li><li>Dillin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rates+of+behavior+and+aging+specified+by+mitochondrial+function+during+development.">Rates of behavior and aging specified by mitochondrial function during development.</a> Science. 298, 2398-2401 (2002).</li><li>Hamilton, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+RNAi+screen+for+longevity+genes+in+C.+elegans.">A systematic RNAi screen for longevity genes in C. elegans.</a> Genes Dev. 19, 1544-1555 (2005).</li><li>Hansen, M., Hsu, A. L., Dillin, A., Kenyon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+genes+tied+to+endocrine,+metabolic,+and+dietary+regulation+of+lifespan+from+a+Caenorhabditis+elegans+genomic+RNAi+screen.">New genes tied to endocrine, metabolic, and dietary regulation of lifespan from a Caenorhabditis elegans genomic RNAi screen.</a> PLoS Genet. 1, 119-128 (2005).</li><li>Lee, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+RNAi+screen+identifies+a+critical+role+for+mitochondria+in+C.+elegans+longevity.">A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity.</a> Nat Genet. 33, 40-48 (2003).</li><li>Smith, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-+and+calorie-independent+life+span+extension+from+dietary+restriction+by+bacterial+deprivation+in+Caenorhabditis+elegans.">Age- and calorie-independent life span extension from dietary restriction by bacterial deprivation in Caenorhabditis elegans.</a> BMC Dev Biol. 8, 49-49 (2008).</li><li>Hodgkin, J., Horvitz, H. R., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nondisjunction+Mutants+of+the+Nematode+CAENORHABDITIS+ELEGANS.">Nondisjunction Mutants of the Nematode CAENORHABDITIS ELEGANS.</a> Genetics. 91, 67-94 (1979).</li><li>Emmons, S. W., Sternberg, P. W., Riddle, D. L., Blumenthal, T., Meyer, B. J., Priess, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. ELEGANS II. , (1997).</li><li>Lipton, J., Kleemann, G., Ghosh, R., Lints, R., Emmons, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mate+searching+in+Caenorhabditis+elegans:+a+genetic+model+for+sex+drive+in+a+simple+invertebrate.">Mate searching in Caenorhabditis elegans: a genetic model for sex drive in a simple invertebrate.</a> J Neurosci. 24, 7427-7434 (2004).</li><li>Davis, B. O., Anderson, G. L., Dusenbery, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+luminescence+spectroscopy+of+fluorescence+changes+during+aging+in+Caenorhabditis+elegans.">Total luminescence spectroscopy of fluorescence changes during aging in Caenorhabditis elegans.</a> Biochemistry. 21, 4089-4095 (1982).</li><li>Klass, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+in+the+nematode+Caenorhabditis+elegans:+major+biological+and+environmental+factors+influencing+life+span.">Aging in the nematode Caenorhabditis elegans: major biological and environmental factors influencing life span.</a> Mech Ageing Dev. 6, 413-429 (1977).</li><li>Steinkraus, K. 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The genetic control of longevity has been studied extensively in C. elegans, largely due to the ease and rapidity with which life span can be determined. The protocol discussed in this article describes a basic framework for obtaining reproducible life span data in C. elegans and can also be applied to relatednematode species.2 By making some simple alterations these procedures can be adapted to measure life span under a variety of conditions. Here we will discuss several common variations, including live bacteria, RNA interference (RNAi), dietary restriction by bacterial deprivation, and drug-free NGM.
Probably the most common variation from this protocol is the maintenance of worms on live bacteria.&nbsp; This can be accomplished by making a few minor changes. First, change the procedure for seeding the plates with bacteria (steps 1.7 through 1.13). Instead of growing OP50 cultures to saturation, grow to mid-log phase and pipette 200 &micro;L onto the plates directly from the liquid culture. Allow the bacteria to grow up on the plates over night and omit exposure to UV. Ampicillin should also be excluded from the plates with FUDR. An advantage to using live bacteria is that worms do not have to be transferred as often to new plates, since bacterial food is alive and growing. The disadvantage of live bacterial food is that OP50 is pathogenic to C. elegans.3 Worms grown on live bacteria have a shorter live span than worms grown on UV-killed bacteria,3 which could potentially mask life span phenotypes associated with aging. Median life span on live bacteria is approximately 20 days.
Gene knock down by RNAi is easily accomplished in C. elegans by modifying their bacterial food so that it produces double-stranded RNA corresponding to the gene to be knocked down. Two RNAi bacterial libraries are available that cover more than 90% of the open reading frames in the C. elegans genome.4-9 To utilize RNAi in the context of life span, replace the OP50 bacteria with the RNAi clone of interest and follow the modifications discussed in the previous paragraph for measuring life span on live bacteria. The RNAi libraries use a plasmid based expression system. The plasmid is selected for using a carbenicillin resistance cassette and expression of the double stranded RNA is induced by isopropyl &beta;-d-thiogalactopyranoside (IPTG). Both carbenicillin and IPTG need to be included in the NGM plates. Modify step 1.4 by adding 100 &micro;L 1 M IPTG and 50 &micro;L 50 mg/mL carbenicillin per 100 mL NGM to both types of plates. Ampicillin does not need to be added to either type of plate, since carbenicillin fulfills the same role.
Dietary restriction is the most widely studied intervention for slowing aging across evolutionarily divergent species. In C. elegans, maximum life span extension on solid media is observed when the bacterial food source is completely removed during adulthood, a form of dietary restriction termed bacterial deprivation (BD) .9, 10 To measure life span in the context of BD, make two modifications to the above protocol. First, prepare a third type of plate. These plates should be identical to the Amp/FUDR plates except lacking the bacterial food source. Second, modify Part 3 to include an additional step between step 3.2 and step 3.3. On the 4th day of adulthood (4 days after transferring the L4 larvae to Amp/FUDR) transfer BD worms to Amp/FUDR plates lacking bacteria. One complication associated with this form of dietary restriction is the worms&rsquo; tendency to flee. In the absence of food the worms will not stay near the center of the plate, but will increase their area of exploration in search of food. As a result, a larger fraction of the worms will crawl up the walls of the plate and desiccate. We often see 50% to 70% of the animals flee after being transferred to BD plates. To address this issue, start with three times as many worms in order to have a significant number remaining on the plates post-flight. BD can also be combined with live bacterial food with no additional modifications, or with RNAi with one additional modification. For BD with RNAi, an additional antibiotic must be added to the plates without bacteria to prevent bacteria transferred with the worms from growing and providing an unwanted food source. Two examples are tetracycline and kanamycin, either of which can be added to the FUDR plates without bacterial food during step 1.4.&nbsp; BD can also be initiated as early as 2 days of adulthood or as late as 14 days of adulthood, with no significant impact on life span.10 
The final modification that we will discuss is measuring life span on NGM plates without additional drugs (e.g. ampicillin or FUDR). This can be accomplished by simply not adding the drugs to the NGM during step 4 and adding one additional step in Part 3. Without FUDR the worms will continue laying eggs and producing larva. During their reproductive phase (approximately the first week of adulthood) all experimental worms will have to be transferred to fresh plates every 2 days to separate them from their offspring.
C. elegans are primarily hermaphroditic with rare occurrence of males. Life span is typically measured for hermaphrodites only, but can also be measured for males. There are two challenges associated with working with male C. elegans. The first is acquiring a large quantity of male worms, as hermaphrodite self-fertilization produces a very small fraction of male offspring (0.1%).11 Once a population containing male worms is attained, male/hermaphrodite mating produces approximately equal numbers of males and hermaphrodites as long as worms are maintained in the presence of food.12 Male stocks can be ordered from the Caenorhabditis Genetics Center or generated by visually screening for founding males produced from hermaphrodite self-fertilization. The second challenge is male scavenging behavior. In the absence of either food or potential mates (hermaphrodites), male worms enter a searching behavioral mode that involves a wide range of movement.13 When maintained on plates this behavior results in a large fraction of the worms fleeing up the plate walls and desiccating. The general method for dealing with this difficulty is simply to start with enough males that a substantial number remain after most have fled. &nbsp;&nbsp;&nbsp;&nbsp;
Apart from life span, a common age-associated phenotype is lipofuscin accumulation. Lipofuscin is complex cellular waste that cannot be degraded that builds up in cells with age and is used as a biomarker of aging in C. elegans.14, 15 Lipofuscin fluoresces and can be easily visualized in C. elegans using the DAPI channel of a fluorescence microscope (Figure 3). Lipofuscin accumulation can be visualized in worms being scored for life span directly on the NGM plates, allowing collection of a useful secondary phenotype in parallel with life span.
In addition to life span, the protocol described in this article can also be used to score phenotypic progression of age-associated paralysis in C. elegans models of proteotoxicity disease.16 When a worm becomes paralyzed it becomes unable to crawl across the plate, but can still move its head. A worm is scored as paralyzed if it fails to move relative to the NGM in response to plate tapping or prodding with a platinum transfer pick, but does move its head. Worms that die typically retain the paralysis score (paralyzed or not paralyzed) that they were given during the most recent live observation. Importantly, even wild type C. elegans become paralyzed with advanced age. For this reason paralysis is typically not scored for worms older than approximately 20 days, as beyond this point it becomes difficult to distinguish between paralysis caused by normal aging and paralysis caused by progression of the proteotoxicity disease.

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<td>Agar</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
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<td>Ampicillin</td>
<td>Reagent</td>
<td>MidSci</td>
<td>0339</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0123-17-3 (211705)</td>
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<td>BactoPeptone</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
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<td>Sigma-Aldrich</td>
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<td>Isopropyl &#x03B2;-d-Thiogalactopyranoside (IPTG)</td>
<td>Reagent</td>
<td>Gold BioTechnology (GBT)</td>
<td>I2481C5</td>
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<td>KPi Dibasic</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>5087862 (3252-01)</td>
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<td>KPi Monobasic</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
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<td>Reagent</td>
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<td>Tris</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>T1503</td>
<td>&nbsp;</td>
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<td>Yeast Extract</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0886-17-0 (288620)</td>
<td>&nbsp;</td>		</tbody>
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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.

Watch this Scientific Journal Video about Measuring Caenorhabditis elegans Life Span on Solid Media at JoVE.com

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.

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 ...

measuring-caenorhabditis-elegans-life-span-on-solid-media

Nanyang Technological University

Research

JoVE Journal

Biology

29.7K Views.  University of Washington. In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Video: Measuring Caenorhabditis elegans Life Span on Solid Media

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 ...

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.

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.

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

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

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 ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

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 ...

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, ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...