Nous remercions Chris Webster pour effectuer les expériences préliminaires de la figure 3 des résultats représentatifs et Jason Watts et Chris Webster des commentaires utiles sur le manuscrit. Le financement de cette étude a été fourni par une subvention des National Institutes of Health (USA) (R01DK074114) à JLW. Certaines souches de nématodes utilisés dans ce travail ont été fournies par le Centre de Génétique Caenorhabditis, qui est financé par le Bureau NIH des programmes d&#39;infrastructure de recherche (P40 de OD010440).

Les acides gras polyinsaturés sont sensibles à la chaleur, la lumière et l&#39;oxygène. Par conséquent, des précautions doivent être prises lors de la préparation de plaques de supplémentation d&#39;acides gras tels que les acides gras ne sont pas exposés à une chaleur excessive et de la lumière. NGM support contenant 0,1% de Tergitol (NP-40) sont passés à l&#39;autoclave et partiellement refroidies, après quoi les sels de sodium d&#39;acides gras sont ajoutés sous agitation constante. Les plaques sont laissées à sécher à l&#39;obscurité. L&#39;absorption des acides gras par C. elegans cultivé sur ces plaques peut alors être surveillé par Chromatographie en phase gazeuse. Une. Préparation des acides gras Supplement médias <ol><li> Mesurer composants multimédia dans un ballon de taille appropriée. Par 1 L, ajouter 17 g de Bacto-agar, 2,5 g de tryptone, 3 g de NaCl, 1 ml 5 mg / ml de cholestérol dissous dans de l&#39;éthanol, et 10 ml de 10% Tergitol dissous dans l&#39;eau. </li><li> Ajouter 80% du volume final souhaité de l&#39;eau Millipore et l&#39;autoclave les médias ainsi que des bouteilles de verre vide (égal au nombre de différentsLes concentrations d&#39;acides gras à tester), ainsi que des éprouvettes graduées appropriées. </li><li> Médias refroidir dans un bain-marie réglé à 55 ° C. Bien que le milieu est refroidit à préparer la solution de travail de réserve de sel de sodium d&#39;acide gras en brisant ouvert le flacon de verre, en utilisant des mesures de sécurité et d&#39;assurer des particules de verre ne se mélangent pas avec les acides gras. Peser suffisamment d&#39;acide gras pour faire un stock de travail 100 mM. </li><li> Porter la solution d&#39;acide gras à une concentration finale de 100 mM avec de l&#39;eau purifiée. Dissoudre entièrement le sel de sodium d&#39;acide gras prend généralement environ 20 à 30 min. Purger le stock de travail d&#39;acide gras avec de l&#39;argon ou de l&#39;azote, parce que le contact avec un gaz inerte empêche l&#39;oxydation. Boucher le flacon et stocker le stock de travail dans l&#39;obscurité jusqu&#39;à ce que les médias aient refroidi. </li><li> (Facultatif) Si ARNi médias est souhaitée, de mesurer l&#39;ampicilline et isopropilene β-D-1-thiogalactopyranoside (IPTG) solutions ici. </li><li> Lorsque la gélose est refroidie à 55 ° C, ajouter par 1 L: 1ml de 1 M MgSO 4, 1 ml de 1 M de CaCl2, et de 25 ml de tampon phosphate (108,3 g KH 2 PO 4 et de 35,6 g de K 2 HPO 4 pour 1 L à l&#39;autoclave). Ajouter le filtre stérilisé ampicilline et solutions IPTG si la fabrication de plaques de RNAi. Pour tous les types de plaques, ajouter de l&#39;eau stérile pour porter le volume de médias final à 1 L. </li><li> Près d&#39;une flamme, transférer des médias à une autoclave et taille appropriée éprouvette graduée puis ajouter de l&#39;eau chaude stérile au volume désiré. Transfert médias retour au flacon initial et mélanger en remuant. </li><li> Près d&#39;une flamme, transférer médias dans un certain nombre de bouteilles égal au nombre de concentrations à tester en mesurant avec un autoclave et de taille appropriée éprouvette graduée. Maintenir les médias aliquotés comme liquide en utilisant un bain d&#39;eau jusqu&#39;à ce que le stock de travail d&#39;acide gras a entièrement dissous. </li><li> Placez une bouteille sur une plaque d&#39;agitation et remuer jusqu&#39;à ce que le support est chaud au toucher, mais pas chaud. Assurez-vous de vous laisser assez detemps à remuer dans la solution mère d&#39;acide gras et versez plaques devant les médias commence à se solidifier. Si la plaque d&#39;agitation a le contrôle de la température, réglez-le à 55 ° C. </li><li> Diluer le stock de travail d&#39;acide gras de 100 mM dans le milieu de la concentration finale souhaitée. médias Remuer jusqu&#39;à ce que le précipité blanc est en solution (environ 1 min). Les médias peuvent encore être légèrement nuageux par la suite. </li><li> (Facultatif) Si l&#39;on souhaite ARNi médias retenir l&#39;ampicilline à 0,1 mg / ml et 2 mM d&#39;IPTG à une concentration finale. </li><li> Verser médias utilisant une aide de la pipette automatique et une aiguille stérile de 25 ml pipette, en ajoutant 8 ml de milieu par 60 mm plaque ou 25 ml de milieu par plaque de 100 mm. Répéter addition d&#39;acide gras et de la plaque de coulée étapes pour les bouteilles restantes. </li><li> Après solidification de la gélose, couvrir les plaques d&#39;acides gras supplémenté avec une boîte ou un magasin à la température ambiante dans un tiroir bien ventilé à l&#39;abri de l&#39;oxydation de la lumière. </li><li> Graine E. coli OP50 sur des plaques, deux jours après les plaques ont séchées.Pour les plaques de 60 mm, une pipette 300 ul d&#39;une culture d&#39;une nuit OP50 (cultivée à 37 ° C). Si les plaques d&#39;ARNi ont été versés, semences avec 300 pi de bactéries ARNi après les plaques ont séché pendant quatre jours. Plate temps de séchage peut être nécessaire de modifier en raison des différences d&#39;humidité dans divers environnements de laboratoire. </li><li> Incuber les plaques dans un environnement sombre à la température ambiante pendant la pelouse bactérienne est le séchage. </li></ol> 2. Induire la destruction des cellules germinales par la supplémentation de DGLA <ol><li> C. elegans cultivées sur des plaques contenant mM DGLA 0,3 (20h03 n-6) deviennent stériles à cause de la destruction des cellules germinales dans les deux nématodes de la scène et adultes larvaires. <ol><li> Préparer une population synchronisée des larves L1 en traitant hermaphrodites gravides avec une solution d&#39;hypochlorite alcalin (pour une solution à 10 ml: 0,5 ml de NaOH 5M, 2,5 ml d&#39;eau de Javel, et 7 ml H 2 O). Secouez délicatement les hermaphrodites gravides dans cette solution jusqu&#39;à ce que le ver adultes dissoudre. Les œufs sont conservés, et peuvent être rassemblées en un culot par centrifugation à faible vitesse. </li><li> Laver la préparation d&#39;oeuf 3 fois dans du tampon M9 (3 g KH 2 PO 4, 6 g de Na 2 HPO 4, 5 g de NaCl, 1 ml 1 M MgSO 4, H 2 O de 1 L. Ajouter MgSO 4, après passage à l&#39;autoclave). Afin de fournir suffisamment d&#39;oxygène, de remettre en suspension les oeufs dans un tube en plastique de 15 ml à un volume final de 5 ml de tampon M9 et de la roche sur un agitateur pendant une nuit. </li><li> Larves L1 doit être ajouté aux plaques supplémenté deux jours après l&#39;ensemencement avec OP50, ou un jour après l&#39;étalement des bactéries d&#39;ARNi. Incuber les vers à 20 ° C pendant trois jours, ou jusqu&#39;à ce qu&#39;ils atteignent le stade adulte. </li></ol></li><li> Les vers adultes peuvent être marqués de la stérilité en utilisant un microscope à dissection avec un grossissement assez haut pour visualiser le développement des embryons dans l&#39;utérus. Succès la perte de cellules germinales apparaît comme un vide de l&#39;utérus clair des œufs. </li><li> Les vers peuvent également être marqués par la fixation et coloration avec l&#39;acide nucléique d&#39;uncid colorant diamindinophenylindole (DAPI), ce qui facilite la visualisation des noyaux dans les cellules germinales et les embryons en développement. Une tache de DAPI rapide est réalisé par la cueillette vers dans une goutte de tampon M9 sur un verre de montre 9. <ol><li> Flood verre de montre avec 1 ml de 0,2 ng / m. DAPI dans une solution d&#39;éthanol à 95%, laisser reposer pendant environ 5 min. </li><li> Choisissez vers dans une goutte de Vectashield sur une lame, puis couvrir avec une lamelle. </li><li> lamelle de Seal et conserver à 4 ° C dans l&#39;obscurité pendant une nuit pour la coloration optimale, mais les diapositives peuvent également être consulté immédiatement. Note pour la présence ou l&#39;absence de noyaux de cellules germinales à l&#39;aide d&#39;un microscope à fluorescence équipé d&#39;une lampe UV et filtre. </li></ol></li></ol> 3. Confirmant acides gras absorption par chromatographie en phase gazeuse Composition de C d&#39;acides gras globale elegans peuvent être déterminées par la production d&#39;esters méthyliques d&#39;acides gras (FAME) qui sont ensuite séparés et quantifiés à l&#39;aide de gaz chromatography 4. <ol><li> Recueillir 500-1000 vers adultes en les lavant hors des plaques avec de l&#39;eau et de transférer à un 13 mm x 100 mm tube supérieur de vis de verre silanisée. </li><li> Laissez vers déposent par gravité, puis retirez autant d&#39;eau que possible avec une pipette Pasteur en verre. </li><li> Laver avec de l&#39;eau une fois vers, puis retirez à nouveau autant d&#39;eau que possible. </li><li> FAMEs sont formées en ajoutant 1 ml de 2,5% de H 2 SO 4 dans le méthanol, puis chauffage à 70 ° C pendant 1 heure dans un bain d&#39;eau. </li><li> Enlever les tubes du bain d&#39;eau et de refroidir pendant 1 min. </li><li> Extrait FAMEs en ajoutant 1,5 ml d&#39;eau et 0,25 ml d&#39;hexane. </li><li> tubes de reboucher et agiter vigoureusement. </li><li> Tubes à centrifuger dans une centrifugeuse clinique de table pendant 1 min à vitesse maximale pour séparer l&#39;hexane de solvant aqueux. </li><li> Transfert hexane (couche supérieure) pour un insert GC flacon dans un flacon de GC, en faisant attention à ne pas transférer de la phase aqueuse. Pour un GCnalyse, 1-2 pi de FAME à l&#39;hexane est injecté sur une colonne de chromatographie en phase gazeuse capillaire polaire adapté pour l&#39;analyse des FAME. Injecteur de GC Agilent 7890 est fixée à 250 ° C, avec un débit de 1,4 ml / min, et le four de GC est programmé pour une température initiale de 130 ° C qui est maintenue pendant 1 min. Par la suite, la température est en rampe de 10 ° C / min jusqu&#39;à 190 ° C, puis de nouveau en rampe à 5 ° C / min jusqu&#39;à 210 ° C et maintenue pendant 1 min. </li><li> Pour faire en sorte que l&#39;absorption de DGLA a eu lieu, l&#39;analyse FAME de détection de ionisation de flamme (FID) ou la spectrométrie de masse (MS) la détection, en utilisant des étalons authentiques pour l&#39;identification de l&#39;C. elegans acides gras. </li></ol>

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Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Ici, nous décrivons une méthode de supplémentation de C. elegans avec des acides gras insaturés alimentaires. Comme mentionné ci-dessus, il faut prendre soin dans la préparation de plaques PUFA supplémenté en raison de la nature réactive des doubles liaisons dans les acides gras polyinsaturés des causes de ces acides gras pour être sensible à l&#39;oxydation par la chaleur et de la lumière 11. Pour éviter l&#39;oxydation, il est important d&#39;ajouter le PUFA dans le milieu de gélose ...

Les acides gras sont des composants structurels essentiels des membranes ainsi que des molécules de stockage d&#39;énergie efficace. En outre, les acides gras peuvent être clivés à partir de membranes cellulaires par des lipases et peuvent être modifiées par voie enzymatique pour produire des effecteurs de signalisation 1. D&#39;origine naturelle des acides gras polyinsaturés (AGPI) contiennent deux ou plusieurs doubles liaisons cis. Les acides gras oméga-3 acides gras et les acides gras oméga-6 se distinguent l&#39;une de l&#39;autre sur la base des positions des doubles liaisons par rapport à l&#39;extrémité méthyle de l&#39;acide gras. Une alimentation saine exigent à la fois acides gras oméga-6 et oméga-3 acides gras. Cependant, les régimes occidentaux sont particulièrement riches en acides gras oméga-6 acides gras et pauvres en oméga-3 acides gras. Une haute teneur en oméga-6 et oméga-3 ratio d&#39;acide gras est associée à un risque accru de maladies cardiovasculaires et inflammatoires, cependant, les fonctions bénéfiques et nuisibles précis d&#39;acides gras spécifiques ne sont pas bien comprises 2. Les ascaris elega Caenorhabditisns est utile pour étudier la fonction des acides gras, car il synthétise toutes les enzymes nécessaires pour produire une gamme d&#39;acides gras oméga-6 et oméga-3 acides gras, y compris une oméga 3 désaturase, une activité qui est absente dans la plupart des animaux 3,4. Mutants dépourvus enzymes désaturase d&#39;acide gras ne parviennent pas à produire des PUFA spécifiques, conduisant à une série de défauts de développement et neurologiques 4-6. Pour étudier les effets physiologiques des acides gras alimentaires, nous avons développé un dosage biochimique compatible avec l&#39;analyse génétique en utilisant les deux techniques knock-down ARNi mutant et C. elegans. La supplémentation avec des PUFA spécifiques est obtenue en ajoutant une solution de sel de sodium d&#39;acide gras dans le milieu d&#39;agar avant la coulée. Cela se traduit par l&#39;absorption par le PUFA E. source de nourriture coli, où il s&#39;accumule dans les membranes bactériennes. C. elegans ingèrent les bactéries contenant des AGPI, et ce complément alimentaire est suffisante pour sauver le défectueuxts de mutants déficients en AGPI. La supplémentation de la plupart des acides gras n&#39;a pas d&#39;effets néfastes sur les animaux de type sauvage, cependant, oméga-6 acides gras spécifiques, en particulier l&#39;acide dihomo-gamma-linolénique (DGLA, 20:03 n-6) causer une destruction permanente de C. elegans cellules germinales 7,8. Chromatographie en phase gazeuse est utilisé pour surveiller l&#39;absorption de l&#39;acide gras complétée dans la source de nourriture bactérienne (soit OP50 ou HT115), ainsi que dans les nématodes. L&#39;addition du Tergitol de détergent (NP-40) dans le support permet d&#39;obtenir une distribution uniforme des acides gras à travers toute la plaque et l&#39;utilisation plus efficace des acides gras par le E. coli et les nématodes. Nous avons découvert que les acides gras insaturés sont facilement absorbés par les bactéries et C. elegans, mais l&#39;absorption d&#39;acides gras saturés est beaucoup moins efficace. Cet article décrit étape par étape comment compléter les médias agar avec des acides gras, ainsi que la façon de contrôler l&#39;absorption des acides gras dans le ee nématode par chromatographie en phase gazeuse.

<table border="0" cellpadding="0" cellspacing="0" width="765">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Bacto-Agar</td>
			<td style="width:128px;">Difco</td>
			<td style="width:155px;">214010</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Tryptone</td>
			<td style="width:128px;">Difco</td>
			<td style="width:155px;">211705</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">NaCl</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3624-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Tergitol</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">NP40S-500mL</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Cholesterol</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">C8667-25G</td>
			<td style="width:275px;">(5 mg/mL in ethanol)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">MgSO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">2504-01</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">CaCl2</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">1311-01</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">K2HPO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3254-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">KH2PO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3246-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sodium dihomogamma linolenate</td>
			<td style="width:128px;">NuCHEK</td>
			<td style="width:155px;">S-1143</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Warm sterile Millipore water</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sterile water for collecting worms</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Nuclease-free Water for DGLA stock solution</td>
			<td style="width:128px;">Ambion</td>
			<td style="width:155px;">AM9932</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Ampicillin</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">BP1760-25</td>
			<td style="width:275px;">100 mg/ml in water (for RNAi plates)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td>
			<td style="width:128px;">Gold Biotechnology</td>
			<td style="width:155px;">12481C100</td>
			<td style="width:275px;">1 M in water (for RNAi plates)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">HSO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">9681-03</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Methanol</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">A452-4</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Hexane</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">H302-4</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">diamindinophenylindole (DAPI)</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">D9542</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">VectaShield</td>
			<td style="width:128px;">Vector Laboratories</td>
			<td style="width:155px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Glass Flask</td>
			<td style="width:128px;">Corning</td>
			<td style="width:155px;">4980-2L</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Autoclaveable Glass bottles with stirbars</td>
			<td style="width:128px;">Fisherbrand</td>
			<td style="width:155px;">FB-800</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Autoclaveable Glass Graduated Cylinder</td>
			<td style="width:128px;">Fisherbrand</td>
			<td style="width:155px;">08-557</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Stir Plate</td>
			<td style="width:128px;">VWR</td>
			<td style="width:155px;">97042-642</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Waterbath at 55+ &#176;C</td>
			<td style="width:128px;">Precision Scientific Inc.</td>
			<td style="width:155px;">66551</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Screwcap Brown Glass Vial</td>
			<td style="width:128px;">Sun SRI</td>
			<td style="width:155px;">200 494</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Argon gas tank</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Automated Pipette aid</td>
			<td style="width:128px;">Pipette-Aid</td>
			<td style="width:155px;">P-90297</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sterile Serological Pipettes (25 ml)</td>
			<td style="width:128px;">Corning</td>
			<td style="width:155px;">4489</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Bunsen Burner</td>
			<td style="width:128px;">VWR</td>
			<td style="width:155px;">89038-534</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Dissection microscope</td>
			<td style="width:128px;">Leica</td>
			<td style="width:155px;">TLB3000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Silanized glass tube</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:155px;">STT-13100-S</td>
			<td>for FAMEs derivitization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">PTFE Screw caps</td>
			<td style="width:128px;">Kimble-Chase</td>
			<td style="width:155px;">1493015D</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Clinical tabletop centrifuge</td>
			<td style="width:128px;">IEC</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Crimp Vial</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Vial Insert</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 232</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Vial cap</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Gas Chromatograph</td>
			<td style="width:128px;">Agilent</td>
			<td style="width:155px;">7890A</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Mass Spectrometry Detector</td>
			<td style="width:128px;">Agilent</td>
			<td style="width:155px;">5975C</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Column for gas chromatography</td>
			<td style="width:128px;">Suppelco</td>
			<td style="width:155px;">SP 2380</td>
			<td>30 m x 0.25 mm fused silica capillary column</td>
		</tr>
	</tbody>
</table>

dietary supplementation of polyunsaturated fatty acids in caenorhabditis elegans

Les acides gras sont essentiels pour de nombreuses fonctions cellulaires. Ils servent molécules de stockage d&#39;énergie efficaces, forment le noyau hydrophobe des membranes, et de participer à diverses voies de signalisation. Caenorhabditis elegans synthétise toutes les enzymes nécessaires pour produire une gamme d&#39;acides gras oméga-6 et oméga-3 acides gras. Ceci, combiné avec la simple anatomie et la gamme d&#39;outils génétiques disponibles, en font un modèle intéressant pour étudier la fonction des acides gras. Afin d&#39;étudier les voies génétiques qui assurent la médiation des effets physiologiques des acides gras alimentaires, nous avons mis au point un procédé pour compléter l&#39;C. alimentation elegans avec des acides gras insaturés. La supplémentation est un moyen efficace pour modifier la composition en acides gras de vers et peut également être utilisé pour sauver les défauts de mutants déficients en acides gras. Notre méthode utilise la croissance à moyen agar nématode (NGM) additionné de sels gras de acidsodium. Les acides gras présents dans les plaques deviennent incorporation complétéested dans les membranes de la source d&#39;alimentation bactérienne, qui est ensuite reprise par le C. elegans qui se nourrissent de bactéries complétées. Nous décrivons également un protocole de Chromatographie en phase gazeuse pour contrôler les changements dans la composition d&#39;acide gras qui se produisent dans les vers complétés. C&#39;est un moyen efficace de compléter les régimes de grandes et de petites populations de C. elegans, permettant une gamme d&#39;applications de cette méthode.

La supplémentation de la C. elegans alimentation est limitée par la capacité de la source d&#39;alimentation à l&#39;absorption de bactéries et d&#39;incorporer des acides gras dans la membrane bactérienne. Pour déterminer la capacité de E. coli OP50 à assimiler différents acides gras dans ses membranes, OP50 a été plaqué sur des supports sans supplément, 0,1 mM et les concentrations mM 0,3 d&#39;acide stéarique (18h00), l&#39;oléate de sodium (18:1 n-9), et le sodium DGLA (20 : 3n-6). ...

Watch this Scientific Journal Video about Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans at JoVE.com

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

&lt;em&gt; Caenorhabditis elegan &lt;/ em&gt; est un modèle utile pour explorer les fonctions d&#39;acides gras polyinsaturés dans le développement et la physiologie. Ce protocole décrit une méthode efficace de compléter la &lt;em&gt; C. elegans &lt;/ em&gt; alimentation avec des acides gras polyinsaturés.

La supplémentation alimentaire en acides gras polyinsaturés dans<em&gt; Caenorhabditis elegans</em

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

dietary-supplementation-polyunsaturated-fatty-acids-caenorhabditis

Research

JoVE Journal

Biology

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

17.0K vues.  Washington State University.  Caenorhabditis elegan est un modèle utile pour explorer les fonctions d'acides gras polyinsaturés dans le développement et la physiologie. Ce protocole décrit une méthode efficace de compléter la C. elegans alimentation avec des acides gras polyinsaturés. 

Vidéo: Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

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 ...

Recherche

Biologie

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 ...

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 ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

Combinée des nucléotides et des Extractions de protéine chez Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

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 ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

Purification et analyse de Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

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.

Quantification du déclin protéostatique spécifique aux tissus chez Caenorhabditis elegans

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

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

Modélisation des maladies neurodégénératives associées à l’âge à Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...

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.

Automatisation de la quantification agrégée chez 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 ...