Questo lavoro è stato sostenuto da un premio pilota dal Centro Shock Nathan (P30 AG13283) e una borsa di R01 (R01 AG028516) dal National Institute on Aging (NIA) di A.-LH

<html> 1. Stabilire una curva di calibrazione per la stima delle concentrazioni di batteri Questa sezione descrive un metodo per la stima della concentrazione di batteri per ogni ceppo dato batteri (ad esempio, OP50, HT115) che verrà utilizzato come fonte di cibo per esperimenti di restrizione dietetica in C. elegans. Curve di calibrazione separati dovrebbero essere stabiliti per ciascun batteri ceppo usato. <ol><li> Inoculare una singola colonia di E. coli OP50 batteri selezionati da una vena fresca di batteri su agar LB in 3 ml di brodo LB liquido. </li><li> Luogo OP50 cultura in 37 ° C shaker e permettere ai batteri di crescere durante la notte. </li><li> Preparare seriale 2-diluizioni della sospensione batterica dalla cultura durante la notte (2x, 4x, 8x, 16x, 32x). </li><li> Misurare l&#39;assorbanza (densità ottica) di ogni diluizione a 600 nm con spettrofotometro in triplice copia. </li><li> Diluire ulteriormente ogni sospensione batterica 1.000.000 volte (1000-diluizione due volte). </li><li> Diffusione 0,4 ml di sospensione di batteri di ogni diluizione in modo uniforme su una piastra di agar LB 100 mm. </li><li> Posizionare le piastre LB con OP50 in un incubatore a 37 ° C per 18-24 ore. </li><li> Contare il numero di colonie batteriche coltivate su ogni piatto. La conta delle colonie vengono utilizzati per calcolare ufc (unità formanti colonia) per ml per ogni campione. </li><li> Il rapporto tra i valori misurati OD 600 e OP50 concentrazioni di batteri può essere stabilita tracciando OD 600 in funzione della concentrazione batterica (ufc / ml) e l&#39;esecuzione di un&#39;analisi di regressione lineare (Figura 1). </li></ol> Soluzioni: <table border="1"><tbody><tr><td colspan="2"> Luria Broth (LB), 1L: </td></tr><tr><td > 10 g </td><td> Bacto-triptone </td></tr><tr><td> 5 g </td><td> Estratto di Lievito </td></tr><tr><td> 10 g </td><td> NaCl </td></tr></tbody></table> Aggiungere acqua deionizzata a 1L. Autoclave e conservare a temperatura ambiente. 2. Preparazione del solido piatto a base di restrizione dietetica (SDR) piastre Questa sezione descrive la preparazione di piatti per l&#39;uso in solido piatto a base di restrizione dietetica (SDR) esperimenti. Normalmente, si considera una concentrazione di batteri di 1 x 10 11 ufc / ml come condizione l&#39;alimentazione ad libitum e 1 x 10 8 ufc / ml come condizione ottimale di alimentazione DR per questo metodo. Tuttavia, 4-6 differenti concentrazioni dei batteri può essere necessario per stabilire una completa relazione dose-dipendente per SDR e le sue risposte. <ol><li> Inoculare una singola colonia di E. Coli OP50 batteri in 3 ml di brodo LB liquido. </li><li> Posizionare il OP50 cultura in 37 ° C shaker e permettere ai batteri di crescere per 6-8 ore. </li><li> Trasferire il OP50 cultura in un pallone nuovo contenente 500 ml di brodo LB, e posizionare il OP50 cultura indietro nel 37 ° C durante la notte shaker. </li><li> Misurare il diametro esterno 600 della cultura OP50 per determinare la concentrazione di batteri (la concentrazione è calcolata utilizzando la curva di calibrazione generata in parte 1). Diluizioni appropriate possono essere necessari per una misurazione accurata di assorbanza. </li><li> Pellet OP50 batteri mediante centrifugazione a 1.500 g per 20 minuti. </li><li> Risospendere batteri a una concentrazione di 1 x 10 12 ufc / ml. </li><li> Preparare 10 mL di coltura batterica in sei diverse concentrazioni: 1 x 10 12, 10 11, 10 10, 10 9, 10 8, 10 7 ufc / ml. </li><li> Pipettare 0,2 ml di coltura batterica al centro di una piastra da 60 mm solidificato NGM. Evitare di toccare la superficie della piastra con puntale, le scalfitture sulla superficie permettono di scavare vermi nei piatti di agar. </li><li> Piastre posto nella 37 ° C incubatore per 1 ora. </li><li> Applicare 10μL di 100mg/ml carbenicillina e 10 ml di kanamicina 50mg/ml ad ogni piatto di arrestare la crescita dei batteri. </li><li> Conservare le piastre con diverse concentrazioni di antibiotico-batteri uccisi a 4 ° C per un uso futuro (fino a un mese). E &#39;importante preparare e conservare le piastre a sufficienza per l&#39;intero esperimento programmato almeno 1-2 giorni a venire. Utilizzando lo stesso lotto di piastre per gli esperimenti intero è preferibile. </li><li> Per verificare la vitalità dei batteri, raschiare alcuni batteri fuori delle piastre SDR e stenderlo su un piatto vuoto con NGM carbenicillina e kanamicina. Posizionare la piastra a 37 ° C incubatore per 6-8 ore e poi controllare per la crescita dei batteri (nessuna crescita dei batteri devono essere osservati). </li></ol> Soluzioni <table border="1"><tbody><tr><td colspan="2"> Nematode crescita Media (NGM), 1L: </td></tr><tr><td > 3 g </td><td> NaCl </td></tr><tr><td> 2,5 g </td><td> Bacto-peptone </td></tr><tr><td> 20g </td><td> Agar </td></tr></tbody></table>ove_content &quot;autoclave&gt; per 40 minuti e lasciare raffreddare a 65 ° C, quindi aggiungere: <table border="1"><tbody><tr><td > 1 ml </td><td> 1 M MgSO 4 </td></tr><tr><td> 1 ml </td><td> 1 M CaCl 2 </td></tr><tr><td> 1 ml </td><td> 5 mg / ml colesterolo </td></tr><tr><td> 25 mL </td><td> Potassio fosfato 1M, pH 6.0 </td></tr></tbody></table> Immediatamente versare NGM liquido sul 60 mm x 15 millimetri capsule di Petri in 10 aliquote mL 3. La creazione di esperimenti DSP Per la maggior parte delle SDR-studi collegati, come l&#39;analisi durata della vita e profilatura fertilità, la preparazione di un età sincronizzato popolazione di vermi è essenziale. Per altri studi, è sufficiente trasferire i vermi da un regolare OP50 piastra per un piatto di SDR di avviare la restrizione dietetica. In questa sezione, un protocollo per impostare un&#39;analisi vita per gli animali SDR è descritto come un esempio. <ol><li> Utilizzare un selettore a vite senza fine di platino per trasferire 20-30 adulti sessualmente attivi su diverse piastre NGM regolari con OP50. A seconda del numero di uova necessarie per gli esperimenti, più adulti possono essere necessari. </li><li> Lasciare le piastre a 20 ° C per 4 ore per consentire la deposizione delle uova. </li><li> Rimuovere gli adulti dal piatto. Piastre deve essere ispezionato visivamente per le uova. </li><li> Lasciare le piastre a 20 ° C e permettono la progenie di sviluppare nella fase 1 adulto Late-L4/day. Di solito, questo richiede 3 giorni per N2 worm selvatici tipi a 20 ° C. </li><li> Trasferire un numero adeguato di giorno 1 adulti sulla piastra SDR di avviare la restrizione dietetica. </li><li> Per un&#39;analisi durata della vita, istituito per un totale di 6-8 piatti con 10-20 vermi per piastra per ogni condizione di SDR. </li><li> Punteggio la vitalità di ogni verme ogni 2 giorni fino a quando tutti i vermi sono morti. E &#39;fondamentale che i vermi essere trasferiti a piastre SDR fresco ogni 12-48 ore per evitare la completa privazione di cibo. </li></ol> 4. Rappresentante dei risultati: Mostrato nella Figura 1 è un grafico di una curva di calibrazione rappresentante per la stima della concentrazione OP50 in relazione al diametro esterno 600. L&#39;equazione generato attraverso l&#39;analisi di regressione rivestimento può essere utilizzato per tutti i futuri calcoli di OP50 concentrazioni di batteri. Illustrato nella figura 2 è un esempio di saggiare l&#39;effetto dei trattamenti DSP cronica durata media di ceppi di vite diverse. La linea blu rappresenta una normale risposta dose-dipendente di tipo selvaggio worm di SDR. La linea rossa indica che l&#39;effetto durata di DSP è parzialmente soppressa dal DRR-2 oe, mentre daf-16 (mu86) non ha effetto su SDR-indotta prolunga la durata della vita. Metodo di dettaglio per l&#39;esecuzione di analisi durata in C. elegans è descritto in letteratura 1-3 precedenti. <img src="/files/ftp_upload/2701/2701fig1.jpg" alt="Figura 1" /> Figura 1. Rappresentante risultato di una curva di calibrazione per la stima di E. Coli OP50 concentrazione. OP50 concentrazione è tracciata in funzione del diametro esterno 600 valori misurati. Le barre di errore = SD cultura Pernottamento batterica è stato diluito 1.5x, 2x, 4x, 8x, 16x, 32x prima misurazione dell&#39;assorbanza, e ulteriormente diluito 1.000 x due volte prima di misurazione cfu. <img src="/files/ftp_upload/2701/2701fig2.jpg" alt="Figura 2" /> Figura 2. Risultato di un rappresentante C. elegans esperimento di vita a confronto animali selvatici tipo N2 (blu) con due diversi mutanti (verde e rosso) in risposta a DSP (Figura da Ching et al. 4). Barre di errore = SEM

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

Restrizione dietetica (DR) è noto per aumentare la durata della vita e ritardare l&#39;insorgenza di varie malattie legate a una vasta gamma di specie, tra cui i mammiferi 5-7. Entrambe le manipolazioni ambientali e genetici sono stati utilizzati per modellare DR in C. elegans, tra cui vermi coltura in un semi-definita liquido 8,9, diluizione della fonte di cibo batterica in terreno liquido 10-12, e l&#39;uso di mutanti comportamenti che alimentano ad un ritmo più lento 1...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Carbenicillina </td><td> Duchefa Biochemie </td><td> C0109.25 </td><td> Disciolto in acqua. </td></tr><tr><td> Kanamicina </td><td> Roche </td><td> 10106801001 </td><td> Disciolto in acqua. </td></tr><tr><td> Bacto-peptone </td><td> Becton, Dickinson and Company </td><td> DF0118072 </td><td></td></tr><tr><td> Bacto-triptone </td><td> Becton, Dickinson and Company </td><td> DF0123075 </td><td></td></tr><tr><td> Bacto-estratto di lievito </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> Cultura provetta sterile </td><td> USA scientifico </td><td> 1485-0810 </td><td></td></tr><tr><td> Spettrofotometro </td><td> Amersham Biosciences </td><td> Gene Quant pro </td><td> Amersham ora fa parte di GE Healthcare. </td></tr><tr><td> Microscopio da dissezione </td><td> Zeiss </td><td> Stemi 2000 </td><td></td></tr><tr><td> Centrifuga </td><td> Beckman Coulter </td><td> Accuspin FR </td><td></td></tr></tbody></table>

solid plate-based dietary restriction in caenorhabditis elegans

Riduzione del consumo di cibo, senza fame o malnutrizione è conosciuto per aumentare la durata della vita e ritardare l&#39;insorgenza di varie malattie legate a una vasta gamma di specie, tra i mammiferi. Essa provoca anche una diminuzione del peso corporeo e fertilità, così come bassi livelli di glicemia, insulina e IGF-1 in questi animali. Questo trattamento è spesso definito come restrizione dietetica (CD) o restrizione calorica (CR). Il nematode Caenorhabditis elegans è emerso come un organismo importante modello per lo studio della biologia dell&#39;invecchiamento. Entrambe le manipolazioni ambientali e genetici sono stati utilizzati per modellare DR e hanno dimostrato di prolungare la durata della vita in C. elegans. Tuttavia, molti degli studi DR riportato in C. elegans sono stati fatti da animali di propagazione in mezzi liquidi, mentre la maggior parte degli studi genetici nel campo dell&#39;invecchiamento sono stati fatti su l&#39;agar standard di solido in piastre di Petri. Qui vi presentiamo un protocollo DR utilizzando le normali solido NGM agar a base di piastra con batteri uccisi.

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

Qui vi presentiamo un protocollo per l&#39;esecuzione di solido piatto a base di restrizioni alimentari,<em&gt; C. elegans</em&gt; Con batteri uccisi.

Solida piastra a base di restrizioni alimentari, 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 ...

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

Research

JoVE Journal

Biology

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

16.5K Views.  University of Michigan. Qui vi presentiamo un protocollo per l'esecuzione di solido piatto a base di restrizioni alimentari, C. elegans Con batteri uccisi.

Video: Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Measuring Caenorhabditis elegans Life Span on Solid Media

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

Measuring Caenorhabditis elegans Life Span on Solid Media

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

Misura Caenorhabditis elegans Life Span su terreni solidi

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

Ricerca

Biologia

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.

Isolamento e In vitro Attivazione di 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 ...

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.

Microscopia time-lapse di Embriogenesi All&#39;inizio del 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.

Di base<em&gt; Caenorhabditis elegans</em&gt; Metodi: sincronizzazione e di osservazione

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.

Prostaglandina estrazione e l&#39;analisi in<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 supplementazione alimentare di acidi grassi polinsaturi a<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.

Misurazione dei tassi di consumo di ossigeno in intatto 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.

Quantificazione del declino proteostatico tessuto-specifico nella 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.

Modellazione di malattie neurodegenerative associate all'età in 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.

Automatizzare la quantificazione aggregata 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 ...