Ce travail a été soutenu par un nouveau chercheur Grant [2014R1A1A1005553] de la National Research Foundation de Corée (NRF) à JIL; et un futur chef de l&#39;Université Yonsei Défi Grant [2015-22-0133] pour JIL

1. Préparer des solutions pour NGT-3D et NGB-3D <ol><li> Préparer les solutions stériles suivantes: 1 L de solution 0,1454 M de NaCl, 1 L de 1 M CaCl 2, 1 L de 1 M MgSO 4, Lysogénie Broth (LB), 1 L de 1 M KPO 4 tampon (108,3 g de KH 2 PO 4 et 35,6 g de K 2 HPO 4 et H 2 O compléter à 1 L). Production de NGM utilisant ces solutions peuvent être trouvées dans un précédent protocole 10. </li><li> Solutions autoclave à 121 ° C, 15 min. </li><li> Préparer 50 ml d&#39;une / ml de solution stérile, 5 mg de cholestérol. Dans un tube conique de 50 ml, mélanger 0,25 g de cholestérol et de 50 ml de 99,99% d&#39;éthanol et bien mélanger. Ne pas autoclaver. Stériliser la solution de cholestérol à l&#39;aide d&#39;une seringue de 50 ml avec un filtre à seringue de 0,45 um. Cela permettra également de supprimer tout le cholestérol non dissous. </li><li> (Facultatif) Préparer 10 ml d&#39;une 150 mM de 2&#39;-désoxy-5-fluorouridine (FUdR) stock. Dans un tube conique de 15 ml, mélanger 0,3693 gde FUdR et 10 ml d&#39;eau distillée. Bien agiter. </li></ol> 2. Préparer les bactéries de la culture pour NGT-3D et NGB-3D <ol><li> Inoculer 10 ml LB bouillon avec des bactéries. Les bactéries standards utilisés pour C. elegans alimentation est la souche E. coli OP50. </li><li> La culture d&#39;inoculation bactérienne à 37 ° C dans un incubateur avec agitation pendant une nuit. </li><li> Dans la préparation d&#39;une dilution en série, aliquotes de 9 ml de la solution de NaCl dans 15 ml tubes coniques stériles. Par exemple, aliquote 9 ml dans un total de 7 tubes pour obtenir une dilution de la souche 10 -7 OP50 E. coli. </li><li> À l&#39;aide d&#39;une pipette stérile 1000 ul, pipette 1 ml de la culture bactérienne ou d&#39;une culture bactérienne diluée dans le nouveau tube stérile avec 9 ml de solution de NaCl et bien vortexer le mélange de 10 ml. Répétez jusqu&#39;à ce que la dilution bactérienne désirée soit atteinte. REMARQUE: La dilution bactérienne souhaitée dépend du type et de l&#39;état des bactéries, ainsi que les conditions expérimentales précises.Par exemple, un 10 -6 - 10 -8 dilution de la souche OP50 de E. coli a été suffisante pour produire à partir de 1 - 200 colonies bactériennes par 6,5 ml Tube NGT-3D. Worms fiable développé et reproduit normalement lorsque le nombre de colonies était de plus de 60, qui a eu lieu à une dilution de 10 -6 à 10 -7. Pour NGB-3D, utiliser une dilution de 10 -8. </li></ol> 3. Faire NGT-3D et NGB-3D (200 ml) <ol><li> Après la préparation de la culture bactérienne, mélanger 0,6 g de NaCl, 1 g de granulé de la gélose et la peptone 0,5 g dans un flacon de 500 ml. Insérez une barre d&#39;agitation magnétique dans le ballon. </li><li> Ajouter 195 ml d&#39;eau distillée et couvrir la bouche de la fiole avec une feuille d&#39;aluminium. </li><li> Autoclave à 121 ° C pendant 15 min. </li><li> Placer le ballon autoclavé chaud sur une plaque d&#39;agitation, et agiter à une vitesse modérée pendant au moins 2 h. Refroidir le ballon à 40 ° C. Assurez-vous de refroidir suffisamment en continuant d&#39;ici à des températures élevées peut conduire à opacification de l&#39;agar fini.Cependant, des températures plus basses peuvent conduire à un durcissement prématuré de la gélose. <ol><li> (Facultatif) Pour accélérer le processus de refroidissement, placer le ballon dans un 40 ° C bain d&#39;eau 15 min avant de le placer sur une plaque d&#39;agitation. </li></ol></li><li> Lorsque la température du milieu gélose atteint 40 ° C, ajouter 200 pi de 1 M CaCl2, 200 ul de 5 mg / ml de solution de cholestérol, 200 pi de 1 M MgSÛ4 et 5 ml de 1 M KPO 4 tampon que la solution continue d&#39;agiter à des concentrations finales de 1 mM de CaCl2, 5 ug / ml de cholestérol, 1 mM de MgSO 4, 1 mM KPO 4. <ol><li> (Facultatif) Pour l&#39; essai de durée de vie NGT-3D ajouter 80 ul de mM FUdR 150 à une concentration finale de 120 uM 12. </li></ol></li><li> Ajouter 6 ml des 10 ml de culture bactérienne diluée à l&#39;étape 2.4 dans le ballon directement. <ol><li> (Facultatif) Pour NGT-3D, retirez 6 ml de milieu agar avant l&#39;étape 3.6 et le garder au chaud séparément. Ce média sera utilisé pour les bactéries librescouche supérieure. </li></ol></li><li> En utilisant des procédures stériles, distribuer les médias dans une chambre de culture stérile. Assurez-vous que le couvercle de la chambre se ferme hermétiquement. <ol><li> (Facultatif) Pour éviter des colonies bactériennes de se former à la surface supérieure de la gélose, faire une couche de bactéries libres-média agar de l&#39;étape 3.6.1 sur le dessus avant que les médias de l&#39;étape 3.7 durcit complètement. Cela va créer une zone exempte de bactéries dans le haut de la NGT-3D. </li><li> Pour NGT-3D, versez 6,5 ml de milieu dans les 8 ml claires tubes à essai en plastique pour faire une couche de gélose bactéries, et distribuer soigneusement 200 pi des médias exempt de bactéries sur le dessus du support semi-durci 3D pour faire une aux bactéries mince couche libre sur le dessus. </li><li> Pour NGB-3D, versez 65 ml de milieu dans les 25 cm 2 plastique transparent flacon de culture cellulaire. Ce montant doit remplir le corps de la 25 cm 2 de culture cellulaire bouteille. </li></ol></li><li> Laissez chambres verticalement à la température ambiante pendant une semaine pour permettre des colonies bactériennes de croîtred&#39;une taille considérable d&#39;au moins 1 mm de diamètre. <ol><li> (Facultatif) Pour l&#39;essai de durée de vie dans NGT-3D, les chambres de couverture avec une feuille d&#39;aluminium pour empêcher la dégradation de la lumière de FUdR. </li></ol></li></ol> 4. Mesurer Fitness de la population de Ver sur NGT-3D (Brood relative Taille Assay) <ol><li> Choisissez un ver de stade L4 en utilisant un fil de platine sélectionner et transfert sur une plaque NGM exempt de bactéries. Laisser un ver de se déplacer librement autour pendant quelques minutes pour éliminer les bactéries attachées à son corps. </li><li> Répétez l&#39;étape 4.1 et assurez-vous que le ver est exempt de bactéries. En règle générale, deux répétitions de 4,1 suffisent. </li><li> Placez délicatement le ver propre sur la surface du support 3D avec un pic de fil de platine. Le ver devrait finalement entrer dans l&#39;agar dans la matrice d&#39;agar 3D. </li><li> Fermez le couvercle permettant lâche un peu d&#39;air d&#39;entrer dans le tube, mais empêchant le séchage du milieu agar. </li><li> Incuber pendant 96 heures à 20 ° C. </li><li> Après 4 jours, fermez le couvercle hermétiquement et placer lechambre de culture NGT-3D dans un bain d&#39;eau C 88 ° pour faire fondre l&#39;agar-agar. La chaleur tue les vers mais leurs corps restent intacts. NOTE: L&#39;utilisation de 8 ml tubes pour NGT-3D, 20 - 30 minutes d&#39;incubation est généralement suffisant. </li><li> En utilisant une pipette en verre, transférer les médias fondus sur un plat en plastique de Pétri de 9 cm. pipettes en plastique ne sont pas informés, que les vers peuvent souvent tenir à eux. </li><li> En utilisant un microscope à dissection transmission stéréo, compter le nombre de vers à la L3, L4 et stades adultes. Ne pas compter les vers qui sont stade L2 et les plus jeunes, les générations F1 et F2 peuvent être confondus ici. Ainsi, cette analyse est une analyse de la taille des couvées par rapport au lieu d&#39;un dosage de la taille totale du couvain. </li></ol> 5. image et enregistrement Worm Comportement sur NGB-3D <ol><li> Choisissez un ver de stade L4 en utilisant un fil de platine sélectionner et éliminer toutes les bactéries collées à la surface par le transfert à une plaque NGM exempt de bactéries et de lui permettre de se déplacer librement pendant quelques minutes. </li><li> Répétez l&#39;étape 5.1 pour vous assurer que le worm est exempt de bactéries. </li><li> Placez délicatement le ver propre sur le centre de la surface de la gélose de la NGB-3D à proximité du col de la bouteille avec un pic de fil de platine. Le ver devrait finalement entrer dans l&#39;agar dans la matrice d&#39;agar 3D. </li><li> Fermez le couvercle permettant lâche un peu d&#39;air d&#39;entrer dans le tube, tout en empêchant le séchage du milieu agar. </li><li> Image ou enregistrer le ver sous un microscope binoculaire transmission stéréo. Réglez la mise au point que le ver se déplace à travers la matrice 3D. </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+local+genetic+diversity+and+low+outcrossing+rate+in+Caenorhabditis+elegans+natural+populations.">High local genetic diversity and low outcrossing rate in Caenorhabditis elegans natural populations.</a> Curr Biol. 15 (13), 1176-1184 (2005).</li><li>Lee, T. Y., Yoon, K. H., Lee, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGT-3D:+a+simple+nematode+cultivation+system+to+study+Caenorhabditis+elegans+biology+in+3D.">NGT-3D: a simple nematode cultivation system to study Caenorhabditis elegans biology in 3D.</a> Biol Open. 5 (4), 529-534 (2016).</li><li>Lewis, J. A., Flemming, J. T., Epstein, H. F., Shakes, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Culture+Methods.">Basic Culture Methods.</a> Caenorhabditis elegans.: Modern Biological Analysis of an Organism. 48, 4-27 (1995).</li><li>Choi, S. Y., Yoon, K. H., Lee, J. I., Mitchell, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Violacein:+Properties+and+Production+of+a+Versatile+Bacterial+Pigment.">Violacein: Properties and Production of a Versatile Bacterial Pigment.</a> Biomed Res Int. , 465056 (2015).</li><li>Solis, G. M., Petrascheck, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Caenorhabditis+elegans+life+span+in+96+well+microtiter+plates.">Measuring Caenorhabditis elegans life span in 96 well microtiter plates.</a> J Vis Exp. (48), (2011).</li><li>Kwon, N., Pyo, J., Lee, S. J., Je, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-D+worm+tracker+for+freely+moving+C.+elegans.">3-D worm tracker for freely moving C. elegans.</a> PLoS One. 8 (2), e57484 (2013).</li><li>Pierce-Shimomura, J. T., Chen, B. L., Mun, J. J., Ho, R., Sarkis, R., McIntire, S. L. <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+crawling+and+swimming+locomotory+patterns+in+C.+elegans.">Genetic analysis of crawling and swimming locomotory patterns in C. elegans.</a> Proc Natl Acad Sci U S A. 105 (52), 20982-20987 (2008).</li><li>Kwon, N., Hwang, A. B., You, Y. J., SJ, V. L., Je, J. 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The authors have nothing to disclose.

La culture en laboratoire de C. elegans en utilisant les plaques de milieu de croissance des nématodes classiques était cruciale pour les centaines de découvertes importantes que la recherche en C. elegans a fourni. Ici, nous présentons de nouvelles méthodes pour cultiver C. elegans dans un environnement qui reflète plus fidèlement leurs habitats naturels en trois dimensions. Bien que d&#39; autres méthodes ont été utilisées pour observer C. elegans 13 en 3D, cec...

The study of the nematode Caenorhabditis elegans in the laboratory has led to seminal discoveries in the field of biology over the last five decades1. C. elegans was the first multicellular organism to have its genome sequenced in 19982, and it has been invaluable in understanding the contributions of individual genes to the development, physiology, and behavior of a whole organism. Scientists now are looking to further understand how these genes may contribute to the survival and reproductive fitness of organisms in their natural environments, asking questions about ecology and evolution at the genetic level3-5. 
C. elegans once again can provide an excellent system to answer these questions. However, little is known about C. elegans biology in natural nematode habitats, and there are no current methods to simulate controlled natural conditions of C. elegans in the laboratory. In the lab, C. elegans is cultivated on the surface of agar plates seeded with E. coli bacteria6. In nature, however, C. elegans and related nematodes can be found sparsely inhabiting soils throughout the globe, but they are specifically found thriving in rotting fruits and vegetative matter7,8. These three-dimensional (3D) complex environments are quite different from the simple 2D environments to which worms are exposed to in the laboratory. 
To begin to answer questions about the biology of nematodes in a more natural 3D setting, we have designed a 3D habitat for laboratory cultivation of nematodes we called Nematode Growth Tube 3D or NGT-3D for short9. The goal was to design a 3D growth system that allows for comparable growth, development, and fertility to the standard 2D Nematode Growth Media (NGM) plates10. This system supports the growth of bacteria and nematodes over their entire life cycles in 3D, allows worms to move and behave freely in three dimensions, and is easy and inexpensive to manufacture and employ. 
In the current study, we provide a step-by-step method to manufacture NGT-3D and evaluate worm development and fertility. In addition to assessing worm fitness in 3D, we sought to image, video, and assess worm behavior and physiology in 3D cultivation. Thus, in addition to NGT-3D, we present here an alternate method called Nematode Growth Bottle 3D or NGB-3D, for the microscopic imaging of C. elegans during 3D cultivation. This will be especially important for the study of known behaviors identified in 2D, and the identification of novel behaviors unique to 3D cultivation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>LB broth, Miller (Luria-Bertani)</td>
			<td>Difco</td>
			<td>224620</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>DAEJUNG</td>
			<td>7548-4400</td>
			<td>58.44 MW</td>
		</tr>
		<tr>
			<td>Agar, Granulated</td>
			<td>Difco</td>
			<td>214530</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Bacto</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride, dihydrate</td>
			<td>Bio Basic</td>
			<td>CD0050</td>
			<td>2*H2O; 147.02 MW</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Bio Basic</td>
			<td>CD0122</td>
			<td>386.67 MW</td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>B&#38;J</td>
			<td>RP090-1</td>
			<td>99.99%; 46.07 MW</td>
		</tr>
		<tr>
			<td>Magnesium sulfate, anhydrous</td>
			<td>Bio Basic</td>
			<td>MN1988</td>
			<td>120.37 MW</td>
		</tr>
		<tr>
			<td>Potassium phosphate, monobasic, anhydrous</td>
			<td>Bio Basic</td>
			<td>PB0445</td>
			<td>136.09 MW</td>
		</tr>
		<tr>
			<td>2&#39;-Deoxy-5-fluorouridine</td>
			<td>Tokyo Chemical Industry</td>
			<td>D2235</td>
			<td>246.19 MW</td>
		</tr>
		<tr>
			<td>Potassium phosphate, dibasic, anhydrous</td>
			<td>Bio Basic</td>
			<td>PB0447</td>
			<td>174.18 MW</td>
		</tr>
		<tr>
			<td>Multi-Purpose Test Tubes</td>
			<td>Stockwell Scientific</td>
			<td>ST.8570</td>
			<td>8 mL</td>
		</tr>
		<tr>
			<td>Test Tube Closures</td>
			<td>Stockwell Scientific</td>
			<td>ST.8575</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Flask</td>
			<td>SPL Lifescience</td>
			<td>70125</td>
			<td>25 cm^2</td>
		</tr>
		<tr>
			<td>Research Stereo Microscope</td>
			<td>Nikon</td>
			<td>SMZ18</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Definition Color Camera Head</td>
			<td>Nikon</td>
			<td>DS-Fi2</td>
			<td></td>
		</tr>
		<tr>
			<td>PC-Based Control Unit</td>
			<td>Nikon</td>
			<td>DS-U3</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements Basic Research, Microscope Imaging Software</td>
			<td>Nikon</td>
			<td>MQS32000</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

La construction de NGT-3D est un protocole simple et direct qui se traduit par un tube d&#39;essai d&#39; agar-rempli avec de petites colonies bactériennes espacées tout au long de l&#39;agar - agar (figure 1A). Les vers peuvent se déplacer librement à travers la matrice d&#39;agar, la recherche et la consommation des colonies bactériennes. Pour vérifier si C. elegans peuvent se reproduire et se développer normalement dans NGT-3D, nous avons comparé la f...

Watch this Scientific Journal Video about Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory at JoVE.com

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

Nous présentons une méthode simple pour construire 3D systèmes de culture de nématodes appelés NGT-3D et NGB-3D. Ceux - ci peuvent être utilisées pour étudier le nématode fitness et des comportements dans des habitats qui sont plus semblables à Caenorhabditis elegans naturelles habitats que les laboratoires C. plaques de culture elegans 2D standard.

la culture de<em&gt; Caenorhabditis elegans</em&gt; En trois dimensions dans le laboratoire

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 ...

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Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

17.7K vues.  Yonsei University.  Nous présentons une méthode simple pour construire 3D systèmes de culture de nématodes appelés NGT-3D et NGB-3D. Ceux - ci peuvent être utilisées pour étudier le nématode fitness et des comportements dans des habitats qui sont plus semblables à Caenorhabditis elegans naturelles habitats que les laboratoires C. plaques de culture elegans 2D standard. 

Vidéo: Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

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.

L&#39;isolement et la In vitro Activation des Caenorhabditis elegans Sperme

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

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Biologie

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.

Mesure Caenorhabditis elegans</emDurée de vie> à 96 plaques de microtitration bien

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.

Solide plaque à base de restriction alimentaire Caenorhabditis elegans</em

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 ...

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.

Time-lapse de microscopie du début de l&#39;embryogenèse dans les 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.

De base<em&gt; Caenorhabditis elegans</em&gt; Méthodes: Synchronisation et d&#39;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 ...

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.

Extraction de prostaglandine et de l&#39;analyse en<em&gt; Caenorhabditis elegans</em

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

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 ...

Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions

The voice of humans and most non-human mammals is generated in the larynx through self-sustaining oscillation of the vocal folds. Direct visual documentation of vocal fold vibration is challenging, particularly in non-human mammals. As an alternative, excised larynx experiments provide the opportunity to investigate vocal fold vibration under controlled physiological and physical conditions. However, the use of a full larynx merely provides a top view of the vocal folds, excluding crucial portions of the oscillating structures from observation during their interaction with aerodynamic forces. This limitation can be overcome by utilizing a hemi-larynx setup where one half of the larynx is mid-sagittally removed, providing both a superior and a lateral view of the remaining vocal fold during self-sustained oscillation.
Here, a step-by-step guide for the anatomical preparation of hemi-laryngeal structures and their mounting on the laboratory bench is given. Exemplary phonation of the hemi-larynx preparation is documented with high-speed video data captured by two synchronized cameras (superior and lateral views), showing three-dimensional vocal fold motion and corresponding time-varying contact area. The documentation of the hemi-larynx setup in this publication will facilitate application and reliable repeatability in experimental research, providing voice scientists with the potential to better understand the biomechanics of voice production.

This paper introduces a protocol for the preparation of hemi-larynx specimens facilitating a multi-dimensional view of vocal fold vibration, in order to investigate various biophysical aspects of voice production in humans and non-human mammals.

Hemi-laryngée Setup pour étudier la Vibration du champ Vocal en trois Dimensions

The voice of humans and most non-human mammals is generated in the larynx through self-sustaining oscillation of the vocal folds. Direct visual ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

Un protocole pour laboratoire logement de Turquoise cyprinodonte (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

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.

Mesure des taux de consommation d’oxygène intactes de Caenorhabditis elegans

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