Ce travail a été soutenu par une bourse de pilote de Centre de choc Nathan (P30 AG13283) et une subvention R01 (R01 AG028516) du National Institute on Aging (NIA) à A.-LH

<html> 1. Établir une courbe d&#39;étalonnage pour l&#39;estimation des concentrations de bactéries Cette section décrit une méthode pour l&#39;estimation de la concentration de bactéries pour toute souche donnée de bactéries (par exemple, OP50, HT115) qui sera utilisé comme source de nourriture pour les expériences de restriction alimentaire en C. elegans. Courbes d&#39;étalonnage distincts devraient être établis pour chaque bactérie souche utilisée. <ol><li> Inoculer une seule colonie de E. coli OP50 bactéries sélectionnées dans une série fraîche de bactéries sur une gélose LB en 3 ml de bouillon LB liquide. </li><li> Placer OP50 culture à 37 ° C shaker et permettent aux bactéries de croître durant la nuit. </li><li> Préparer série 2 dilutions de la suspension bactérienne de la culture de la nuit (2x, 4x, 8x, 16x, 32x). </li><li> Mesurer l&#39;absorbance (densité optique) de chaque dilution à 600 nm par un spectrophotomètre, en trois exemplaires. </li><li> Diluer chaque suspension bactérienne 1000000 fois (1000-dilution deux fois). </li><li> Fais 0,4 ml de suspension bactérienne de chaque dilution de répartir uniformément sur une plaque de 100 mm LB agar. </li><li> Placez les plaques LB avec OP50 dans un incubateur à 37 ° C pendant 18-24 heures. </li><li> Comptez le nombre de colonies bactériennes cultivées sur chaque assiette. Le nombre de colonies sont utilisées pour calculer ufc (unité formant colonie) par ml pour chaque échantillon. </li><li> La relation entre la DO mesurées 600 valeurs et OP50 concentrations de bactéries peuvent être établies en traçant DO 600 en fonction de la concentration bactérienne (UFC / ml) et en effectuant une analyse de régression linéaire (figure 1). </li></ol> Solutions: <table border="1"><tbody><tr><td colspan="2"> Luria Broth (LB), 1L: </td></tr><tr><td > 10 g </td><td> Bacto-tryptone </td></tr><tr><td> 5 g </td><td> Extrait de levure </td></tr><tr><td> 10 g </td><td> NaCl </td></tr></tbody></table> Ajouter de l&#39;eau déminéralisée pour 1L. Autoclave et conserver à température ambiante. 2. Préparation de la plaque solide basée sur la restriction alimentaire (DTS) plaques Cette section décrit la préparation des plaques pour une utilisation en milieu solide plaque de base alimentaires de restriction (DTS) expériences. Normalement, on considère une concentration de bactéries de 1 x 10 11 ufc / mL que l&#39;état du ad libitum d&#39;alimentation et 1 x 10 8 ufc / mL que l&#39;état DR alimentation optimale pour cette méthode. Toutefois, 4-6 différentes concentrations de bactéries peuvent être nécessaires pour établir une complète relation dose-dépendant pour les DTS et ses réponses. <ol><li> Inoculer une seule colonie de E. Coli OP50 bactéries dans 3 ml de bouillon LB liquide. </li><li> Placer la culture dans 37 OP50 agitateur ° C et permettent aux bactéries de croître pendant 6-8 heures. </li><li> Transfert de la culture OP50 à un nouveau flacon contenant 500 ml de bouillon LB, et placer la culture OP50 de retour à 37 ° C durant la nuit agitateur. </li><li> Mesurer la DO 600 de la culture OP50 pour déterminer la concentration des bactéries (la concentration est calculée en utilisant la courbe de calibration générée dans la partie 1). Des dilutions appropriées peuvent être nécessaires pour une mesure précise de l&#39;absorbance. </li><li> Pellet OP50 bactéries par centrifugation à 1500 g pendant 20 minutes. </li><li> Resuspendre bactérie à une concentration de 1 x 10 12 ufc / mL. </li><li> Préparer 10 ml de culture de bactéries dans six concentrations différentes: 1 x 10 12, 10 11, 10 10, 10 9, 10 8, 10 7 ufc / mL. </li><li> Pipeter 0,2 ml de culture de bactéries dans le centre d&#39;une plaque de 60 mm solidifié NGM. Évitez de toucher la surface de la plaque avec la pointe de pipette, que les entailles à la surface permettent de creuser dans les vers de la gélose. </li><li> Placer les plaques dans 37 incubateur ° C pendant 1 heure. </li><li> Appliquer 10 pi de 100mg/mL carbénicilline et 10 uL de kanamycine 50MG/ML à chaque plaque d&#39;arrêter la croissance de la bactérie. </li><li> Plaques magasin avec différentes concentrations d&#39;antibiotiques ont tué les bactéries à 4 ° C pour une utilisation future (jusqu&#39;à un mois). Il est important de préparer et de stocker assez d&#39;assiettes pour l&#39;expérience entière planifié au moins 1-2 jours à venir. En utilisant le même lot de plaques pour les expériences de l&#39;ensemble est préférable. </li><li> Pour vérifier la viabilité de la bactérie, gratter quelques bactéries sur des plaques de DTS et l&#39;étaler sur une plaque de NGM vide avec carbénicilline et la kanamycine. Placer la plaque dans 37 incubateur ° C pendant 6-8 heures puis vérifiez à la croissance des bactéries (pas de croissance des bactéries doit être observé). </li></ol> Solutions <table border="1"><tbody><tr><td colspan="2"> Croissance nématode médias (NGM), 1L: </td></tr><tr><td > 3 g </td><td> NaCl </td></tr><tr><td> 2,5 g </td><td> Bacto-peptone </td></tr><tr><td> 20g </td><td> Agar </td></tr></tbody></table>ove_content &quot;autoclave&gt; pendant 40 minutes et laisser refroidir à 65 ° C, puis ajouter: <table border="1"><tbody><tr><td > 1 ml </td><td> 1 M MgSO 4 </td></tr><tr><td> 1 ml </td><td> 1 M de CaCl 2 </td></tr><tr><td> 1 ml </td><td> 5 mg / ml de cholestérol </td></tr><tr><td> 25 ml </td><td> Phosphate de potassium 1M, pH 6,0 </td></tr></tbody></table> Verser immédiatement NGM liquide sur 60 mm x 15 mm dans les boîtes de Pétri aliquotes de 10 ml 3. Mise en place d&#39;expériences de DTS Pour la plupart des études SDR-connexes, telles que l&#39;analyse la longévité et la fertilité de profilage, la préparation d&#39;un âge synchronisée de la population de vers de terre est essentielle. Pour d&#39;autres études, il suffit de transférer les vers d&#39;une plaque régulière OP50 à une plaque de DTS pour lancer la restriction alimentaire. Dans cette section, un protocole pour mettre en place une analyse durée de vie pour les animaux de DTS est décrit comme un exemple. <ol><li> Utilisez un sélecteur de ver de platine pour le transfert de 20 à 30 adultes reproducteurs actifs sur plusieurs plaques NGM régulier avec OP50. Selon le nombre d&#39;œufs requis pour les expériences, plus d&#39;adultes peut être nécessaire. </li><li> Laisser les plaques à 20 ° C pendant 4 heures pour permettre la ponte des œufs. </li><li> Retirez les adultes à partir de la plaque. Les plaques doivent être inspectés visuellement pour les oeufs. </li><li> Laisser les plaques à 20 ° C et laisser la descendance de développer dans la phase Late-L4/day adulte 1. Habituellement, cela prend 3 jours pour les vers N2 types sauvages à 20 ° C. </li><li> Transfert d&#39;un nombre approprié de jours 1 adulte sur la plaque de DTS pour lancer la restriction alimentaire. </li><li> Pour une analyse cycle de vie, mis en place un total de 6-8 plaques avec 10-20 ver par plaque pour chaque condition de DTS. </li><li> Score à la viabilité de chaque ver tous les 2 jours jusqu&#39;à ce que tous les vers sont morts. Il est essentiel que les vers soient transférés à des plaques de DTS frais tous les 12-48 heures pour éviter la privation complète d&#39;aliments. </li></ol> 4. Les résultats représentatifs: Montre la figure 1 est un tracé d&#39;une courbe d&#39;étalonnage représentant pour l&#39;estimation de la concentration OP50 par rapport à OD 600. L&#39;équation générée par analyse de régression revêtement peut être utilisé pour tous les futurs calculs des concentrations de bactéries OP50. Montre la figure 2 est un exemple de dosage de l&#39;effet des traitements chroniques sur les DTS durée de vie moyenne de souches différentes ver. La ligne bleue représente une normale dose-dépendante de réponse de vers de type sauvage de DTS. La ligne rouge indique que l&#39;effet durée de vie de DTS est partiellement supprimée par la RRC-2 OE, tandis que daf-16 (mu86) n&#39;a aucun effet sur ​​l&#39;extension DTS-induite durée de vie. Détails de la méthode pour effectuer des analyses durée de vie en C. elegans est décrite dans la littérature antérieure 1-3. <img src="/files/ftp_upload/2701/2701fig1.jpg" alt="Figure 1" /> Figure 1. Résultat représentatif d&#39;une courbe de calibration pour l&#39;estimation de E. Coli OP50 concentration. OP50 concentration est tracée en fonction de la mesure OD 600 valeurs. Les barres d&#39;erreur = DD culture de nuit bactérienne a été dilué 1.5x, 2x, 4x, 8x, 16x, 32x avant la mesure d&#39;absorbance, puis dilué 1000 x deux fois avant de mesure UFC. <img src="/files/ftp_upload/2701/2701fig2.jpg" alt="Figure 2" /> Figure 2. Résultat représentatif d&#39;un C. elegans expérience comparant la durée de vie des animaux sauvages type N2 (en bleu) avec deux mutants différents (vert et rouge) en réponse à de DTS (figure de Ching et al. 4). Les barres d&#39;erreur = SEM

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La restriction alimentaire (DR) est connu pour augmenter la durée de vie et de retarder l&#39;apparition de diverses maladies liées au vieillissement dans une large gamme d&#39;espèces, y compris les mammifères 5-7. Manipulations environnementaux et génétiques ont été utilisées pour modéliser DR en C. elegans, y compris les vers de culture dans un milieu semi-défini de liquide 8,9, la dilution de la source de nourriture des bactéries en milieu liquide 10-12, et l&#39...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Carbénicilline </td><td> Duchefa Biochemie </td><td> C0109.25 </td><td> Dissous dans l&#39;eau. </td></tr><tr><td> Kanamycine </td><td> Roche </td><td> 10106801001 </td><td> Dissous dans l&#39;eau. </td></tr><tr><td> Bacto-peptone </td><td> Becton, Dickinson and Company </td><td> DF0118072 </td><td></td></tr><tr><td> Bacto-tryptone </td><td> Becton, Dickinson and Company </td><td> DF0123075 </td><td></td></tr><tr><td> Bacto-extrait de levure </td><td> Becton, Dickinson and Company </td><td> DF0127071 </td><td></td></tr><tr><td> Agar </td><td> Becton, Dickinson and Company </td><td> DF00145170 </td><td></td></tr><tr><td> Tube de culture stérile </td><td> USA Scientific </td><td> 1485-0810 </td><td></td></tr><tr><td> Spectrophotomètre </td><td> Amersham Biosciences </td><td> Gene Quant Pro </td><td> Amersham fait maintenant partie de GE Healthcare. </td></tr><tr><td> Microscope à dissection </td><td> Zeiss </td><td> Stemi 2000 </td><td></td></tr><tr><td> Centrifugeuse </td><td> Beckman Coulter </td><td> Accuspin FR </td><td></td></tr></tbody></table>

solid plate-based dietary restriction in caenorhabditis elegans

Réduction de la consommation d&#39;aliments sans malnutrition ou de famine est connue pour augmenter la durée de vie et de retarder l&#39;apparition de diverses maladies liées au vieillissement dans une large gamme d&#39;espèces, y compris les mammifères. Elle entraîne également une diminution du poids corporel et de la fertilité, ainsi que des niveaux plus bas de glucose plasmatique, l&#39;insuline et l&#39;IGF-1 chez ces animaux. Ce traitement est souvent désigné comme la restriction alimentaire (DR) ou de la restriction calorique (RC). Le nématode Caenorhabditis elegans est apparu comme un organisme modèle important pour étudier la biologie du vieillissement. Manipulations environnementaux et génétiques ont été utilisées pour modéliser DR et ont montré de prolonger la durée de vie en C. elegans. Cependant, la plupart des études ont rapporté DR en C. elegans ont été réalisées en propageant des animaux dans des milieux liquides, tandis que la plupart des études génétiques dans le domaine du vieillissement ont été réalisés sur la gélose standard de solide dans des boîtes de Pétri. Nous présentons ici un protocole standard de DR à l&#39;aide NGM solides à base de gélose plaque avec des bactéries tuées.

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

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Nous présentons ici un protocole pour la réalisation solide plaque de base de la restriction alimentaire au<em&gt; C. elegans</em&gt; Avec des bactéries tuées.

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

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

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Solid Plate-based Dietary Restriction in Caenorhabditis elegans

16.5K vues.  University of Michigan. Nous présentons ici un protocole pour la réalisation solide plaque de base de la restriction alimentaire au C. elegans Avec des bactéries tuées.

Vidéo: Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Measuring Caenorhabditis elegans Life Span on Solid Media

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

Measuring Caenorhabditis elegans Life Span on Solid Media

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

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 ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

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

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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

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

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

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

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

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

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

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

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

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

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

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

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, ...

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 ...

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 ...