This work was supported by a pilot award from the Nathan Shock Center (P30 AG13283) and a R01 grant (R01 AG028516) from the National Institute on Aging (NIA) to A.-L.H.

1. Establishing a calibration curve for the estimation of bacteria concentrations
This section describes a method for the estimation of bacteria concentration for any given bacteria strain (e.g. OP50, HT115) that will be used as a food source for Dietary restriction experiments in C. elegans. Separate calibration curves should be established for each bacteria strain used. 
<ol>
 <li>Inoculate a single colony of E. coli OP50 bacteria picked from a fresh streak of bacteria on LB agar in 3 mL liquid LB broth.</li>
 <li>Place OP50 culture in 37 &deg;C shaker and allow bacteria to grow overnight. </li>
 <li>Prepare serial 2-fold dilutions of the bacterial suspension from the overnight culture (2x, 4x, 8x, 16x, 32x).</li>
 <li>Measure the absorbance (optical density) of each dilution at 600 nm by spectrophotometer in triplicate.</li>
 <li>Further dilute each bacterial suspension 1,000,000-fold (1000-fold dilution twice).</li>
 <li>Spread 0.4 mL of bacterial suspension of each dilution evenly on a 100 mm LB agar plate.</li>
 <li>Place LB plates with OP50 in a 37 &deg;C incubator for 18-24 hours.</li>
 <li>Count the number of bacterial colonies grown on each plate. The colony counts are used to calculate cfu (colony forming unit) per mL for each sample.</li>
 <li>The relationship between measured OD600 values and OP50 bacteria concentrations can be established by plotting OD600 as a function of bacterial concentration (cfu/mL) and performing a linear regression analysis (Figure 1).</li>
</ol>
 Solutions:
<table border="1">
 <tbody>
 <tr>
 <td colspan="2">Luria Broth (LB), 1L:</td>
 </tr>
 <tr>
 <td >10 g</td>
 <td>Bacto-tryptone</td>
 </tr>
 <tr>
 <td>5 g </td>
 <td>Yeast Extract</td>
 </tr>
 <tr>
 <td>10 g</td>
 <td>NaCl</td>
 </tr>
 </tbody>
</table>
Add deionized water to 1L. 
 Autoclave and store at room temperature. 
2. Preparation of solid plate-based dietary restriction (sDR) plates
This section describes the preparation of plates for use in solid-plate based dietary restriction (sDR) experiments. Normally, we consider a bacteria concentration of 1 x 1011 cfu/mL as the ad libitum feeding condition and 1 x 108 cfu/mL as the optimal DR feeding condition for this method. However, 4-6 different bacteria concentrations may be needed to establish a complete dose-dependent relationship for sDR and its responses.
<ol>
 <li>Inoculate a single colony of E. Coli OP50 bacteria in 3 mL liquid LB broth.</li>
 <li>Place the OP50 culture in 37 &deg;C shaker and allow the bacteria to grow for 6-8 hours.</li>
 <li>Transfer the OP50 culture to a new flask containing 500 mL LB broth, and place the OP50 culture back in 37 &deg;C shaker overnight. </li>
 <li>Measure the OD600 of the OP50 culture to determine the bacteria concentration (the concentration is calculated using the calibration curve generated in Part 1). Appropriate dilutions may be needed for an accurate measurement of absorbance. </li>
 <li>Pellet OP50 bacteria by centrifugation at 1,500 g for 20 minutes.</li>
 <li>Resuspend bacteria to a concentration of 1 x 1012 cfu/mL.</li>
 <li>Prepare 10 mL bacteria culture in six different concentrations: 1 x 1012, 1011, 1010, 109, 108, 107 cfu/mL.</li>
 <li>Pipette 0.2 mL of bacteria culture into the center of a 60 mm solidified NGM plate. Avoid touching the surface of the plate with pipette tip, as nicks on the surface allow worms to burrow into the agar plates.</li>
 <li>Place plates in 37 &deg;C incubator for 1 hour.</li>
 <li>Apply 10&mu;L of 100mg/mL carbenicillin and 10 &mu;L of 50mg/mL kanamycin to each plate to arrest growth of the bacteria. </li>
 <li>Store plates with different concentrations of antibiotic-killed bacteria at 4 &deg;C for future use (up to one month). It is important to prepare and store enough plates for the entire planned experiment at least 1-2 days ahead. Using the same batch of plates for the entire experiments is preferable.</li>
 <li>To verify the viability of the bacteria, scrape some bacteria out of the sDR plates and spread it on an empty NGM plate with carbenicillin and kanamycin. Place the plate in 37 &deg;C incubator for 6-8 hours and then check for growth of the bacteria (no bacteria growth should be observed).</li>
</ol>
Solutions
<table border="1">
 <tbody>
 	<tr>
 <td colspan="2">Nematode Growth Media (NGM), 1L:</td>
 </tr>
 <tr>
 <td >3 g </td>
 <td>NaCl</td>
 </tr>
 <tr>
 <td>2.5 g</td>
 <td>Bacto-Peptone </td>
 </tr>
 <tr>
 <td>20g</td>
 <td>Agar</td>
 </tr>
 </tbody>
</table>
Autoclave for 40 minutes and let cool to 65 &deg;C, then add:
<table border="1">
 <tbody>
 <tr>
 <td >1 mL </td>
 <td>1 M MgSO4</td>
 </tr>
 <tr>
 <td>1 mL</td>
 <td>1 M CaCl2</td>
 </tr>
 <tr>
 <td>1 mL</td>
 <td>5 mg/mL Cholesterol</td>
 </tr>
 <tr>
 <td>25 mL</td>
 <td>1M Potassium Phosphate, pH 6.0</td>
 </tr>
 </tbody>
</table>
Immediately pour liquid NGM onto 60 mm x 15 mm petri dishes in 10 mL aliquots
3. Setting up sDR experiments
For most of the sDR-related studies, such as life span analysis and fertility profiling, preparation of an age-synchronized population of worms is essential. For other studies, simply transfer worms from a regular OP50 plate to an sDR plate to initiate the dietary restriction. In this section, a protocol to set up a lifespan analysis for sDR animals is described as an example. 
<ol>
 <li>Use a platinum worm picker to transfer 20-30 reproductively active adults onto several regular NGM plates with OP50. Depending on the number of eggs required for the experiments, more adults may be needed.</li>
 <li>Leave the plates at 20&deg;C for 4 hours to allow egg-laying.</li>
 <li>Remove the adults from the plate. Plates should be visually inspected for eggs.</li>
 <li>Leave the plates at 20&deg;C and allow the progeny to develop into the Late-L4/day 1 adult stage. Usually, this takes 3 days for N2 wild types worms at 20 &deg;C.</li>
 <li>Transfer a suitable number of day 1 adults onto sDR plate to initiate the dietary restriction. </li>
 <li>For a lifespan analysis, set up a total of 6-8 plates with 10-20 worms per plate for each sDR condition. </li>
 <li>Score the viability of each worm every 2 days until all the worms have died. It is critical that worms be transferred to fresh sDR plates every 12-48 hours to avoid complete deprivation of food. </li>
</ol>
4. Representative Results:
Shown in Figure 1 is a plot of a representative calibration curve for the estimation of OP50 concentration in relation to OD600. The equation generated by liner regression analysis can be used for all future calculations of OP50 bacteria concentrations. Shown in Figure 2 is an example of assaying the effect of chronic sDR treatments on mean lifespan of different worm strains. The blue line represents a normal dose-dependent response of wild-type worms to sDR. The red line indicates that the lifespan effect of sDR is partially suppressed by drr-2 o.e., while daf-16(mu86) has no effect on sDR-induced lifespan extension. Detail method for performing lifespan analysis in C. elegans is described in previous literature 1-3.
<img src="/files/ftp_upload/2701/2701fig1.jpg" alt="Figure 1" /> 
Figure 1. Representative result of a calibration curve for the estimation of E. Coli OP50 concentration. OP50 concentration is plotted as a function of measured OD600 values. Error bars = S.D. Overnight bacterial culture was diluted 1.5x, 2x, 4x, 8x, 16x, 32x before absorbance measurement, and further diluted 1,000x twice before cfu measurement.
<img src="/files/ftp_upload/2701/2701fig2.jpg" alt="Figure 2" /> 
Figure 2. Representative result of a C. elegans lifespan experiment comparing wild type N2 animals (blue) with two different mutants (green and red) in response to sDR (Figure from Ching et al. 4). Error bars = S.E.M.

<ol>
	<li>Hsu, A. L., Murphy, C. T., 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=Regulation+of+aging+and+age-related+disease+by+DAF-16+and+heat-shock+factor.">Regulation of aging and age-related disease by DAF-16 and heat-shock factor.</a> Science. 300, 1142-1145 (2003).</li><li>Kenyon, C., Chang, J., Gensch, E., Rudner, A., Tabtiang, 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+mutant+that+lives+twice+as+long+as+wild+type.">A C. elegans mutant that lives twice as long as wild type.</a> Nature. 366, 461-464 (1993).</li><li>Apfeld, J., 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=Regulation+of+lifespan+by+sensory+perception+in+Caenorhabditis+elegans.">Regulation of lifespan by sensory perception in Caenorhabditis elegans.</a> Nature. 402, 804-809 (1999).</li><li>Ching, T. T., Paal, A. B., Mehta, A., Zhong, L., Hsu, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=drr-2+encodes+an+eIF4H+that+acts+downstream+of+TOR+in+diet-restriction-induced+longevity+of+C.+elegans.">drr-2 encodes an eIF4H that acts downstream of TOR in diet-restriction-induced longevity of C. elegans.</a> Aging Cell. 9, 545-557 (2010).</li><li>Weindruch, R., Walford, 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> The Retardation of Aging and Disease by Dietary Restriction. , (1988).</li><li>Masoro, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subfield+history:+caloric+restriction,+slowing+aging,+and+extending+life.">Subfield history: caloric restriction, slowing aging, and extending life.</a> Sci Aging Knowledge Environ. , RE2-RE2 (2003).</li><li>Masoro, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+caloric+restriction+and+ageing.">Overview of caloric restriction and ageing.</a> Mech Ageing Dev. 126, 913-922 (2005).</li><li>Houthoofd, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axenic+growth+up-regulates+mass-specific+metabolic+rate,+stress+resistance,+and+extends+life+span+in+Caenorhabditis+elegans.">Axenic growth up-regulates mass-specific metabolic rate, stress resistance, and extends life span in Caenorhabditis elegans.</a> Exp Gerontol. 37, 1371-1378 (2002).</li><li>Houthoofd, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+reduction+of+metabolic+rate+in+food+restricted+Caenorhabditis+elegans.">No reduction of metabolic rate in food restricted Caenorhabditis elegans.</a> Exp Gerontol. 37, 1359-1369 (2002).</li><li>Klass, M. 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H., Wolff, S., Aguilaniu, H., Durieux, J., 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=PHA-4/Foxa+mediates+diet-restriction-induced+longevity+of+C.+elegans.">PHA-4/Foxa mediates diet-restriction-induced longevity of C. elegans.</a> Nature. 447, 550-555 (2007).</li><li>Lakowski, B., Hekimi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+caloric+restriction+in+Caenorhabditis+elegans.">The genetics of caloric restriction in Caenorhabditis elegans.</a> Proc Natl Acad Sci U S A. 95, 13091-13096 (1998).</li><li>Kaeberlein, T. 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No conflicts of interest declared.

Dietary restriction (DR) is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals 5-7. Both environmental and genetic manipulations have been used to model DR in C. elegans, including culturing worms in a semi-defined liquid medium 8,9, dilution of the bacterial food source in liquid medium 10-12, and use of behavior mutants that feed at a slower rate 13. However, genetic mimics of DR may produce ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>Duchefa Biochemie</td>
			<td>C0109.25</td>
			<td>Dissolved in water.</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Roche</td>
			<td>10106801001</td>
			<td>Dissolved in water.</td>
		</tr>
		<tr>
			<td>Bacto-peptone</td>
			<td>Becton, Dickinson and company</td>
			<td>DF0118072</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bacto-tryptone</td>
			<td>Becton, Dickinson and company</td>
			<td>DF0123075</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bacto-yeast extract</td>
			<td>Becton, Dickinson and company</td>
			<td>DF0127071</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Becton, Dickinson and company</td>
			<td>DF00145170</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile culture tube</td>
			<td>USA Scientific</td>
			<td>1485-0810</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Amersham Biosciences</td>
			<td>Gene Quant pro</td>
			<td>Amersham is now part of GE Healthcare.</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Accuspin FR</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Solid Plate-based Dietary Restriction in Caenorhabditis elegans at JoVE.com

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.

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 ...

solid-plate-based-dietary-restriction-in-caenorhabditis-elegans

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16.5K vistas.  University of Michigan. Aquí se presenta un protocolo para la realización de placa sólida basada en la restricción dietética en C. elegans Con bacterias muertas.

Video: Solid Plate-based Dietary Restriction in Caenorhabditis elegans

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 ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

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 ...

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 ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

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 ...