Nous reconnaissons le Centre Caenorhabditis génétique de C. souches elegans. Le soutien financier a été fourni par l&#39;Institut de recherche Terry Fox. Nous reconnaissons également le soutien accordé aux EP de la Rolande et Marcel Gosselin Graduate stagiaire et la subvention de formation des IRSC / FRSQ en FRN53888 de recherche sur le cancer de l&#39;Université McGill Programme intégré de formation recherche sur le cancer.

1. Préparation des réactifs <ol><li> Préparation des médias pour C. la croissance elegans (dans ce cas, les animaux de type sauvage et les animaux FLCN-1 (ok975) mutantes). <ol><li> Préparez modification Seulement Bacto-peptone (MYOB) mélange sec de Youngren contenant 5,5 g de Tris HCl, 2,4 g de base Tris, 31 g de Bactopetone, 20 g de NaCl et 0,08 g de cholestérol. Mélangez bien avec agitation. NOTE: Ce mélange est suffisante pour préparer 10 L de milieu MYOB. REMARQUE: les plaques normales milieu de croissance (NGM) pourraient être utilisés à la place des plaques MYOB 27. Cependant, le même type de plaques doit être utilisé pour des comparaisons valables entre les souches et entre les répétitions indépendantes. Dans ce protocole, nous avons seulement utilisé des plaques de MYOB. </li><li> Préparer 1 L de milieu MYOB en ajoutant 6 g de MYOB mélange sec, 17 g d&#39;agar-agar et porter à 1 L avec de H 2 O et autoclave pendant 45 min à 122 ° C. Verser plaques et laisser sécher à température ambiante pendant 2 jours. </li><li> En utilisant une technique stérile, inoculer 100 ml de culture de milieu de bouillon LB avec E. bactéries coli OP50 et poussent O / N à 37 ° C. </li><li> Plaques semences MYOB avec E. bactéries coli OP50. Lui permettre de grandir à température ambiante pendant 48 h. </li></ol></li><li> Préparation pour la régulation négative des gènes en utilisant l&#39;ARNi <ol><li> Préparer MYOB plaques ARNi-alimentation. Procédez comme dans les étapes 1.1.1 et 1.1.2. Après autoclavage, laisser le milieu se refroidir à environ 55 ° C et ajouter 1 ml de 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) et 1 ml d&#39;ampicilline 100 mM. </li><li> Streak E. coli HT115 bactéries transformées avec un plasmide qui exprime soit de contrôle ou EV-1 FLCN ARNi spécifique à l&#39;ampicilline (50 ug / ml de) / tétracycline (15 ug / ml) des plaques de gélose. Développer la culture O / N à 37 ° C. </li><li> Prélever des colonies et l&#39;inoculer E. coli HT115 bactéries transformées avec un plasmide qui exprime soit EV de contrôle ou FLCN-1 ARNi spécifique et grandissent dans la culture LB / ampicilline (50 ug / ml) à moyen et O / N à 37 &amp; #176; C. </li><li> plaques de semences avec E. coli HT115 HT115 bactéries ou EV-1 FLCN bactéries ARNi. REMARQUE: Conserver les plaques 48 heures à température ambiante avant l&#39;utilisation. Si ARNi knockdown par l&#39;alimentation est efficace, utiliser d&#39;autres méthodes de livraison du ds-ARN 28. </li></ol></li><li> Induisant un stress oxydatif utilisant paraquat (PQ): <ol><li> Préparer du tampon M9 en mélangeant 3 g de KH 2 PO 4, 6 g de Na 2 HPO 4, 5 g de NaCl et 0,25 g de MgSO 4 · 7H 2 O. Apportez jusqu&#39;à 1 L avec de l&#39;eau distillée. La solution en autoclave pendant 45 min à 122 ° C et de stocker des bouteilles à la température ambiante. </li><li> Le jour du dosage, préparer la solution M9 / PQ contenant. Pour ce protocole, utilisez mM PQ 100 (0.1028 g de PQ dissous dans 4 ml de tampon M9 remplit toute une plaques à 96 puits avec 40 pi de M9 / PQ par puits). ATTENTION: PQ est un composé dangereux et très toxique. Poignée selon des directives appropriées. REMARQUE: Lors de l&#39;exécution d&#39;abord un nouveau strain ou une nouvelle condition, il est recommandé de doser la survie dans des concentrations croissantes de PQ afin de déterminer la meilleure concentration à utiliser dans le but de tuer des animaux dans les 7-10 h. </li></ol></li></ol> 2. Préparation de l&#39;âge de la population synchronisée L4 <ol><li> Transfert 20 hermaphrodites adultes gravides à une plaque de MYOB frais ensemencée avec E. bactéries coli OP50. Préparer plusieurs plaques sur la base du nombre de vers nécessaires au cours de l&#39;essai. </li><li> Permettez-leur de pondre des œufs pendant 6 heures, puis en utilisant un ver fil de platine pointe, retirez les mères. Vérifiez les plaques au microscope pour évaluer le nombre d&#39;œufs pondus. </li><li> Laisser les œufs éclosent et se développent à 20 ° C pendant 48 heures. A cette époque, les animaux sont dans un / stade jeune adulte fin L4. Choisissez mêmes animaux de scène et de transférer aux puits contenant de la solution-PQ. REMARQUE: Depuis 48 heures d&#39;incubation applique uniquement aux mutants qui se développent de manière similaire aux animaux de type sauvage, il est important de contrôler le développementtaux de mutants nouvellement testés pour vous assurer qu&#39;elle est conforme à la vitesse de développement de type sauvage, sinon, d&#39;autres échéances doivent être utilisés. </li><li> Sinon, utilisez des techniques de synchronisation. Générer population synchronisée de L1 vers de larves en utilisant une solution d&#39;hypochlorite (25 ml d&#39;eau, 125 ml d&#39;hypochlorite de sodium à 5,25%, 50 ml de NaOH 4; envelopper bouteille avec du papier d&#39;aluminium pour isoler de la lumière). Cependant, pour cet essai, il est déconseillé, car le blanchiment intense peut affecter le comportement des animaux au cours de l&#39;essai. ATTENTION: Manipuler solution d&#39;hypochlorite selon les directives. REMARQUE: Lors de l&#39;utilisation d&#39;ARNi pour réguler à la baisse le gène d&#39;intérêt, synchroniser les hermaphrodites comme décrit dans l&#39;article 2, à l&#39;exception du transfert des mères à plaques ARNi ensemencés avec des bactéries spécifiques ARNi. Lorsque l&#39;effet de choc avec l&#39;ARNi est faible, l&#39;alimentation pour plusieurs générations est recommandé. </li></ol> 3. Effectuer le stress oxydatif Résistance Assay &lt;/ P&gt; <ol><li> Pipette 40 ul de la solution mM PQ 100 (préparé frais) à chaque puits d&#39;une plaque de 96 puits. Utilisez au moins 12 puits comme réplique de chaque état ​​(traitement, mutant, etc.). Utiliser un ver choix de fil de platine, transférer 5-8 animaux de larves L4 à chaque puits indiquant l&#39;heure de début et de la fin des temps. </li><li> Lors du démarrage d&#39;une autre condition, noter l&#39;heure de début et de la fin des temps. Placer la plaque à 20 ° C dans le temps d&#39;incubation entre les transférer vers et marquant animaux morts une heure plus tard à partir de l&#39;heure de début. </li><li> Utilisation de la loupe binoculaire, compter survie à chaque heure. Secouer doucement la plaque avant de commencer à compter. Indiquez les vers qui ne se déplacent pas, même après avoir repéré une lumière à haute intensité, comme des animaux morts. Veuillez également indiquer le nombre d&#39;animaux vivants. NOTE: En regardant la forme de ver, queues et le mouvement de la tête à fort grossissement, aider à déterminer vers morts de vie. </li><li> Répétez cette étape toutes les heures jusqu&#39;à la plupart des vers sont morts. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 4. Détermination de la survie Pourcentage au point Every Time <ol><li> Calculer le nombre total d&#39;animaux par état en ajoutant le nombre total d&#39;animaux transférés à chaque puits. Ignorer les animaux qui sont endommagés ou tués lors d&#39;un transfert. </li><li> Pour chaque point de temps, calculer le nombre total d&#39;animaux morts par condition (somme des animaux morts dans les 12 puits). Pour chaque point de temps, calculer le pourcentage de décès (nombre d&#39;animaux morts divisé par le nombre total d&#39;animaux et multiplié par 100) et le pourcentage de survie (100 pour cent moins décès). </li><li> Répétez ce test au moins 3 fois indépendants. </li></ol>

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The authors declare that they have no competing financial interests.

C. elegans est un organisme modèle attrayant pour étudier la résistance au stress oxydatif in vivo génétiquement car il peut être facilement cultivées, rapidement et conduit à un grand nombre de descendance génétiquement identique. De multiples méthodes pour mesurer la résistance au stress oxydatif ont été précédemment décrit et ils sont basés sur la supplémentation des plaques de culture avec diverses sources telles que les ROS PQ, la roténone, H 2 O 2, et jug...

A reference was corrected in the publication of <a href="http://www.jove.com/video/52746">Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates</a>. There was an error with reference 34 in the Citations section. It has been updated from:
Allen, E., Walters, I.B., Hanahan, D. Brivanib a dual FGF/VEGF inhibitor,is active both first and second line againstmouse pancreatic neuroendocrine tumors developing adaptive/evasive resistance toVEGF inhibition. Clin Cancer Res. 17, (16) 5299-310 (2011).
to:
Allen, A. T., Maher, K. N., Wani, K. A., Betts, K. E., &#38; Chase, D. L. Coexpressed D1- and D2-like dopamine receptors antagonistically modulate acetylcholine release in Caenorhabditis elegans. Genetics. 188, 579-590 (2011).

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

Chez les eucaryotes, la phosphorylation oxydative de prendre place dans la chaîne de transport des électrons des mitochondries est le principal moteur de la production d&#39;énergie sous forme d&#39;ATP. Espèces réactives de l&#39;oxygène (ROS) sont un sous-produit naturel de ce processus. En dépit de leur rôle important en tant que molécules de signalisation, ROS excessive peut conduire à des lésions de l&#39;ADN, des protéines carbonylation, et l&#39;oxydation des lipides. Un déséquilibre entre la production de ROS et de désintoxication provoque le stress oxydatif, qui conduit à l&#39;épuisement de l&#39;énergie, des dommages cellulaires, et déclenche la mort cellulaire 1,2. Le stress oxydatif contribue au vieillissement et au développement de nombreuses maladies mortelles telles que le cancer, le diabète, les maladies cardiovasculaires et neurodégénératives 3-9. Les cellules ont développé des stratégies enzymatiques et non enzymatiques défense pour maintenir les niveaux de ROS approprié et pour protéger leurs électeurs contre les dommages oxydatifs 1,2. La superoxyde dismutase (SOD) enzymes agissent premier à convert superoxyde de H 2 O 2, qui est ensuite transformé en eau par la catalase ou peroxydase enzymes. Non stratégies de défense enzymatiques comprennent principalement des molécules qui réagissent rapidement avec ROS par rapport aux macromolécules cellulaires, protégeant les composants cellulaires essentiels. Malgré le rôle protecteur de ROS enzymes de détoxification, certaines molécules ROS échapper aux mécanismes de défense antioxydants et conduisent à des dommages oxydatifs. Détection, la réparation, et la dégradation des composants cellulaires endommagés sont les stratégies de défense essentielles pendant le stress oxydatif 1,2. Les voies de signalisation impliquées dans la résistance au stress et le stress oxydatif spécifiquement sont très évolutif conservée 10,11. Contrairement à des expériences de culture de cellules où les conditions organismales ne sont que partiellement reproduit, l&#39;étude du stress oxydatif dans des organismes modèles 12,13 a une grande signification. C. elegans est un nématode vivant en liberté qui peut être facilement et économiquement culturés sur une pelouse bactérienne sur les médias agar. Il est de petite taille (environ 1 mm de longueur) et normalement se développe comme un hermaphrodite auto-fertilisation, ce qui facilite les manipulations génétiques. Il a un cycle de vie rapide et une forte capacité de reproduction, produisant environ 300 descendants par génération, ce qui en fait un outil puissant pour effectuer écrans génétiques à grande échelle 14. Les C. elegans génome est entièrement séquencé et 40-50% des gènes sont prévu pour être homologues de gènes associés à des maladies humaines 15-18. Le knock-down de gènes d&#39;intérêt en utilisant l&#39;ARNi est rapide et facile en C. elegans. Gene régulation vers le bas peut être obtenu par l&#39;alimentation des animaux de E. colibacilles qui hébergent un plasmide exprimant l&#39;ARN double brin qui cible l&#39;ARNm d&#39;intérêt 19. Par conséquent, la détermination de la fonction des gènes en utilisant des écrans ARNi à grande échelle a un grand impact sur ​​la compréhension des maladies humaines, y compris le cancer 20,21. Les études de orésistance au stress xidative en C. elegans ont conduit à l&#39;identification de mécanismes conservées de résistance au stress oxydatif 13,22. Certaines filières identifiées sont des voies communes qui modulent la longévité et la résistance à d&#39;autres contraintes ainsi comme l&#39;hypoxie, la chaleur et le stress osmotique. Ces voies comprennent la signalisation de l&#39;insuline, la signalisation TOR, et autophagie. Autres voies clés impliquent la détoxification des ROS tels que des enzymes superoxyde dismutase et de la catalase enzymes, ou la réparation des dommages tels que le choc de la chaleur et de protéines chaperonnes 11,13,22. Ce protocole décrit la façon de déterminer la résistance au stress oxydatif de C. elegans dans un liquide. Nous avons utilisé FLCN-1 (ok975) et les animaux de type sauvage pour démontrer le protocole puisque nous avons précédemment montré une résistance accrue au stress oxydatif sur la perte de FLCN-1 (ok975) en C. elegans 23. Nous avons également montré que cette augmentation de la résistance dépendsur l&#39;AMPK et de l&#39;autophagie, un axe de signalisation qui améliore bioénergétique cellulaire et favorise la résistance au stress 23. PQ est un facteur de stress oxydatif qui interfère avec la chaîne de transport d&#39;électrons pour produire des espèces réactives de l&#39;oxygène 24. Le même dosage peut être adapté et d&#39;autres sources de ROS ou des composés générant des ROS peut être utilisé tel que H 2 O 2 et la roténone. Des essais similaires ont été développées sur des plaques où de faibles concentrations de PQ sont utilisées 25,26. L&#39;avantage de ce test est qu&#39;il est très rapide, et les résultats pourraient être obtenus en une seule journée. En outre, le volume total de liquide utilisé pour effectuer l&#39;essai de résistance au stress oxydatif dans des plaques 96 puits est faible par rapport au volume utilisé pour préparer des plaques contenant PQ. Par conséquent, la quantité de la PQ est utilisé dans le dosage du liquide est faible, ce qui rend le test peu coûteux et limite la production de déchets toxiques. Toutefois, les limites de ce dosage par rapport à des essais de plaque comprennent la lack de la nourriture liquide dans le dosage et la plus faible concentration d&#39;oxygène à l&#39;état liquide par rapport à l&#39;air. Ce sont des facteurs importants qui, dans certains cas, pourraient influencer les résultats. Par conséquent, confirmant reproductibilité en utilisant d&#39;autres méthodes de résistance au stress oxydatif est recommandé de soutenir les résultats obtenus dans cet essai.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Agar bacteriological grade</td>
			<td>Multicell</td>
			<td>800-010-LG</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriological peptone</td>
			<td>Oxoid</td>
			<td>LP0037</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride biotechnology grade</td>
			<td>Bioshop</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma</td>
			<td>C8503-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure tris hydrochloride</td>
			<td>Invitrogen</td>
			<td>15506-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris aminomethane</td>
			<td>Bio Basic Canada Inc</td>
			<td>77-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-202185A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Bioshop</td>
			<td>69-52-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Bio Basic Inc.</td>
			<td>8013-01-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl viologen dichloride hydrate</td>
			<td>Aldrich chemistry</td>
			<td>856177-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 60x15mm</td>
			<td>Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet 10ml</td>
			<td>Fisher</td>
			<td>1367520</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>G-Biosciences</td>
			<td>RC-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma</td>
			<td>M-5921</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Bioshop</td>
			<td>7558-79-4</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Discovery v8 stereo zeiss microscope</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 well clear microtiter plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>flcn-1 RNAi source</td>
			<td colspan="2">Ahringer Library</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring oxidative stress resistance of caenorhabditis elegans in 96-well microtiter plates

Le stress oxydatif, qui est le résultat d&#39;un déséquilibre entre la production et la détoxication des espèces réactives de l&#39;oxygène, est un contributeur majeur à des troubles humaines chroniques, y compris les maladies cardiovasculaires et neurodégénératives, le diabète, le vieillissement et le cancer. Par conséquent, il est important d&#39;étudier le stress oxydatif, non seulement dans des systèmes cellulaires, mais aussi en utilisant des organismes entiers. C. elegans est un organisme modèle attractif pour étudier la génétique de l&#39;oxydation des voies de transduction de signaux de stress, qui sont hautement évolutives conservées. Ici, nous fournissons un protocole pour mesurer la résistance au stress oxydatif dans C. elegans dans un liquide. Brièvement réactifs, ROS induisant tels que le paraquat (PQ) et H 2 O 2 sont dissous dans du tampon M9, et des solutions sont aliquotées dans les puits d&#39;une plaque de microtitrage à 96 puits. L4 synchronisée / jeune adulte C. elegans animaux sont transférés dans les puits (5-8 animaux / puits) et on mesure la survietoutes les heures jusqu&#39;à la plupart des vers sont morts. Lors de l&#39;exécution d&#39;un essai de résistance au stress oxydatif en utilisant une faible concentration de facteurs de stress dans les plaques, le vieillissement pourrait influencer le comportement des animaux sur le stress oxydatif, qui pourraient conduire à une interprétation erronée des données. Cependant, dans l&#39;essai décrit ci-après, ce problème est peu probable étant donné que seules L4 / jeunes animaux adultes sont utilisés. En outre, ce protocole est peu coûteux et les résultats sont obtenus en une seule journée, ce qui rend cette technique attrayante pour les écrans génétiques. Dans l&#39;ensemble, cela va aider à comprendre oxydatives voies de signalisation du stress de transduction, qui pourraient se traduire en une meilleure caractérisation des troubles d&#39;oxydation humaines stress associé.

De type sauvage comparant C. elegans à FLCN-1 (ok975) Les animaux mutants Ici nous avons utilisé PQ 100 mM pour déterminer la résistance de type sauvage C. elegans animaux par rapport à FLCN-1 (ok975) qui a été montré pour résister au stress oxydatif, la chaleur, et l&#39;anoxie 23. Après 4 heures de traitement, 48,3% de type sauvage survécu par rapport à 77,8% de survie dans FLCN-1 (ok975) animaux. Comme prévu, FLCN-1 ...

Watch this Scientific Journal Video about Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates at JoVE.com

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.

Mesurer le stress oxydatif Résistance<em&gt; Caenorhabditis elegans</em&gt; En 96 plaques de microtitration bien

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

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

15.4K vues.  McGill University. 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. 

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

Measuring Caenorhabditis elegans Life Span on Solid Media

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

Measuring Caenorhabditis elegans Life Span on Solid Media

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

Mesure Caenorhabditis elegans</emDurée de vie> sur des milieux solides

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

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Biologie

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 ...

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 ...

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 ...

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

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

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

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

Ice-Cap: un procédé de croissance Arabidopsis Et plants de tomates dans des plaques à 96 puits pour génotypage à haut débit

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

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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

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, ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

Analyse du stress oxydatif dans embryons de poissons zèbres

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

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.

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 ...

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 ...

Mesurer le stress oxydatif Résistance

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Extraction de prostaglandine et de l'analyse en

Analyse du stress oxydatif dans embryons de poissons zèbres

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