Ringraziamo S. Taneja-Bageshwar, K. Beifuss, S. Kedroske, e HC. Hsiao per le discussioni utili. Questo lavoro è stato finanziato da fondi di start-up del Dipartimento TAMHSC di Medicina Molecolare e Cellulare. L&#39;ambito complesso e disco rotante sono stati acquistati con i fondi messi a disposizione dal Dipartimento e l&#39;Ufficio TAMHSC del Decano. Alcuni ceppi sono stati forniti dalla genetica Caenorhabditis Center, che è finanziato dal National Center for Research Resources. pRF4 (rol-6 (su1006)) era un dono del Fuoco A..

1. Preparazione del Dii macchia <ol><li> Preparare una soluzione madre di 20 mg / mL DII (Biotium, Inc., Hayward, CA) in DMF. DII è sensibile alla luce, in modo da proteggere i Dii dalla luce, avvolgendo in un foglio. </li><li> Creare una diluizione di lavoro di Dii con l&#39;aggiunta di 0,6 microlitri magazzino DII a 399,4 microlitri M9 per ogni popolazione. Questo dovrebbe dare una diluizione finale di lavoro di 30 mcg / ml Dii a M9. Questo può essere scalata per la colorazione popolazioni contemporaneamente. Dii scudo dalla luce, avvolgendo il tubo (s) in un foglio. </li></ol> 2. Preparazione dei nematodi <ol><li> Utilizzare una piastra di 60 millimetri che contiene una popolazione di nematodi incontaminata. Lavare gli animali da lastra con una soluzione di 0,5% Triton X-100 nel buffer M9 rimestando delicatamente liquido in un movimento circolare sulla superficie della piastra per sciogliere tutti gli animali larvali e adulti. Trasferimento lavaggio in un tubo sterile 1,5 ml. </li><li> Immediatamente spin down gli animali a 2000 rpm per 30 sec. Rimuovere e gettare il più supernatant possibile senza disturbare la massa di animali sul fondo della provetta. </li><li> Per ridurre il residuo Triton X-100, lavare gli animali utilizzando M9 tampone, rotazione, e rimuovere il surnatante. Ripetere questa operazione una sola volta. </li><li> Aggiungere 400 ml di soluzione di lavoro DII in M9 al tubo e mescolare brevemente nel vortex per sospendere di nuovo gli animali nella soluzione. </li><li> Agitare tubo a 20 ° C in orizzontale per 3 ore a 350 giri al minuto in una luce-ambiente protetto. Se lo si desidera, gli animali possono essere incubate fino a 16 ore per la colorazione. </li><li> Per ridurre la quantità di colorante, spin down gli animali a 2000 rpm per 20 sec. Rimuovere ed eliminare il più possibile surnatante senza disturbare la massa degli animali. </li><li> Animali risospendere in 400 microlitri di buffer M9 e versare il liquido su una porzione priva di batteri di una piastra di agar NGM seminato con OP50 E. coli. Consentire agli animali di recuperare almeno 30 minuti. Durante il tempo di recupero gli animali devono strisciare via dal liquido colorazione DII e sul cibo. Questo passoriduce la fluorescenza di fondo da DII libero. </li></ol> 3. Montaggio e osservando i campioni <ol><li> Sciogliete il 4% agar-agar in acqua con autoclave o un forno a microonde. </li><li> Crea distanziali riutilizzabili, che può essere utilizzato per garantire spessore uniforme del pad agar, con stratificazione due pezzi di nastro di laboratorio su un vetrino. Fai due diapositive distanziatore totale. </li><li> Organizzare un vetrino pulito di vetro tra due diapositive distanziatore. Pipettare circa 150 microlitri (quattro gocce) di agar fuso 4% sul centro del vetrino pulito. Rapidamente coprire l&#39;agar fuso con un ulteriore scivolo di formare un pad agar. Rimuovere con cautela il vetrino di copertura, mantenendo il pad centrato sulla parte superiore della slitta di montaggio. </li><li> Pipettare circa 5 ml di anestetico nematode (100 mM - 1 levamisolo mM, per esempio) sul pad. </li><li> Monte 8-12 animali in anestesia e coprire con un coprioggetto microscopio. </li><li> Osservare gli animali usando un microscopio confocale o dotati di almeno un obietti 40x<html> ve e un DsRed / TRITC (o compatibili) del filtro. La massima eccitazione di fluorescenza di DII è 549 nm e il suo massimo di emissione è 565 nm per il colorante legato (Biotium, Inc., Hayward, CA). </li></ol> 4. Rappresentante Risultati Macchie Dii la cuticola del wild-type e mutanti C. elegans. La superficie cuticolare contiene annuli separate da solchi circonferenziali e, in alcuni stadi, creste longitudinali chiamati ali. Ogni fase di sviluppo ha strutture cuticolare con composizioni diverse 2. Creste o solchi dei due alae e annuli macchia fluorescente, a seconda della composizione della superficie, in tutta stadi larvali e adulte e rimangono visibili fino ad un giorno dopo il recupero con questo metodo. Macchioline di fondo fluorescente sono a volte osservati (figure 2F, G), ma non di routine (Figura 1, Figura 2A-E, H, I). Tutte le immagini sono state scattate con disco rotante o confocale, quando nota, widefield (wf) microscopio composto. ENT &quot;&gt; <img alt="Figura 1" src="/files/ftp_upload/3362/3362fig1.jpg" /> Figura 1. DII macchie cuticola e neuroni ambientalmente esposti. L2 stadio animale. Ingrandimento 630x. Parti dell&#39;immagine mosaico sono stati catturati utilizzando ANNULLAMENTO-Mac software (Tecnologie BioVision, Exton, PA). Le immagini sono state unite con Adobe Photoshop CS3 (Adobe Systems, Inc., San Jose, CA). Barra di scala = 10 micron. DII anche fluorescente macchie amphid e fasmide neuroni sensoriali della testa e la coda rispettivamente (frecce indicano alcuni). <img alt="Figura 2" src="/files/ftp_upload/3362/3362fig2.jpg" /> Figura 2. Dii macchie fluorescenti C. cuticola elegans in tutte le fasi della post-embrionale di sviluppo. Ingrandimento 630x. Barra di scala = 10 micron. La colorazione di tipo selvaggio animali in A) L1; B) L2; C) dauer; D) L3; E) L4, e F) Ciclo di adulto. Le dorsali ali e anulare sono macchiati fluorescente negli animali L1 e dauer (A, C). Dii macchie dorsali anulare negli animali L2(B). Anulare solchi macchia di L3 e L4 animali (D, E). I solchi delle ali e annuli sono macchiati negli animali adulti (FH). Ali sono composti da due, cinque, o tre creste (in L1, dauer o animali adulti, rispettivamente) che corrono la lunghezza dell&#39;animale (frecce) 16. Annuli creare creste circonferenziale intorno all&#39;animale (punte di freccia, F e J). La cuticola di adulti animali mutanti visualizzare moderati difetti organizzazione cuticolare (GH). G) Creste di alae sono discontinui (wf). H) creste ali soprannumerari sono fusi e ramificati o biforcato (wf). Mutanti del gene del collagene presentano ali e difetti dell&#39;organizzazione anulare (J). I) In animali transgenici sovraesprimono pRF4 (rol-6 (su1006)), creste di alae giacciono in un angolo della lunghezza dell&#39;animale. J) Annuli in animali transgenici iperespressione pRF4 (rol-6 (su1006)) mostra un pattern irregolare. <img alt="Figura 3" src="/files/ftp_upload/3362/3362fig3.jpg" /> Figura 3. ExternAl strutture morfologiche sono illuminati dalla colorazione DII. ingrandimento 630x. Barre di scala = 10 micron. Dii evidenzia anche altre caratteristiche esterne, tra cui A) adulti ermafroditi vulva, B) raggi coda maschio adulto e la ventola, e C) ermafrodita punta della coda (biforcuta in questo contesto mutante). <img alt="Figura 4" src="/files/ftp_upload/3362/3362fig4.jpg" /> Cuticola Figura 4. Richiede più tempo per macchiare con DII di neuroni ambientalmente esposti. Ingrandimento 630x. Barra di scala = 10 micron. Dopo due ore di colorazione, amphid (non mostrato) e fasmide neuroni sensoriali (frecce) sono abbastanza colorati. Al contrario, la cuticola dei giovani animali è solo parzialmente macchiato in patch (punta di freccia). <table border="1"><tbody><tr><td> Lavare </td><td> Soluzione di macchia (+ DII) </td><td> Tempo di incubazione </td><td> Cuticola macchiato </td></tr><tr><td> M9 + 0,5% TrIton X-100 </td><td> M9 + 0,5% Triton X-100 </td><td> 2 ore </td><td> no </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> M9 + 0,5% Triton X-100 </td><td> 3 ore </td><td> no </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> M9 </td><td> 2 ore </td><td> parziale </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> M9 </td><td> 3 ore </td><td> sì </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> H 2 0 </td><td> 2 ore </td><td> parziale </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> H 2 0 </td><td> 3 ore </td><td> sì </td></tr></tbody></table> Tabella 1. Colorazione cuticolare in condizioni diverse. Soluzioni diverse e tempi di incubazione sono stati testati per ottimizzare la colorazione cuticolare negli animali. H 2 0, acqua distillata sterile. Colorazione parziale indica colorazione irregolare della cuticola larvale (Figura 4), anche se le macchie cuticola adulti consistently.

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+actin+filaments+in+patterning+the+Caenorhabditis+elegans+cuticle.">The role of actin filaments in patterning the Caenorhabditis elegans cuticle.</a> Dev. Biol. 184, 373-384 (1997).</li><li>Sapio, M. R., Hilliard, M. A., Cermola, M., Favre, R., Bazzicalupo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zona+Pellucida+domain+containing+proteins,+CUT-1,+CUT-3+and+CUT-5,+play+essential+roles+in+the+development+of+the+larval+alae+in+Caenorhabditis+elegans.">The Zona Pellucida domain containing proteins, CUT-1, CUT-3 and CUT-5, play essential roles in the development of the larval alae in Caenorhabditis elegans.</a> Dev. Biol.. 282, 231-245 (2005).</li><li>Johnstone, I. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+sets+of+interacting+collagens+form+functionally+distinct+substructures+within+a+Caenorhabditis+elegans+extracellular+matrix.">Two sets of interacting collagens form functionally distinct substructures within a Caenorhabditis elegans extracellular matrix.</a> Molecular Biology of the Cell. 14, 1366-1378 (2003).</li><li>Link, C. D., Ehrenfels, C. W., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+expression+of+male+copulatory+bursa+surface+markers+in+Caenorhabditis+elegans.">Mutant expression of male copulatory bursa surface markers in Caenorhabditis elegans.</a> Development. 103, 485-495 (1988).</li><li>Tong, Y. G., Burglin, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditions+for+dye-filling+of+sensory+neurons+in+Caenorhabditis+elegans.">Conditions for dye-filling of sensory neurons in Caenorhabditis elegans.</a> J. Neurosci. Methods. 188, 58-61 (2010).</li><li>Singh, R. N., Sulston, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+observations+on+moulting+in+Caenorhabditis+elegans.">Some observations on moulting in Caenorhabditis elegans.</a> Nematologica. 24, 63-71 (1978).</li><li>Collet, J., Spike, C. A., Lundquist, E. A., Shaw, J. E., Herman, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+osm-6,+a+gene+that+affects+sensory+cilium+structure+and+sensory+neuron+function+in+Caenorhabditis+elegans.">Analysis of osm-6, a gene that affects sensory cilium structure and sensory neuron function in Caenorhabditis elegans.</a> Genetics. 148, 187-200 (1998).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li></ol>

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Il metodo di colorazione DII qui presentata permette un modo relativamente veloce e conveniente per visualizzare la cuticola in C. elegans. Da riproporre e ottimizzare un metodo comunemente usato per l&#39;immagine dell&#39;ambiente neuroni sensoriali esposti 15,17, DII può essere usato per macchiare fluorescente sia ali e le strutture anulare (Figure 1 e 2), così come la vulva, la coda di sesso maschile e ermafrodita coda picco (Figura 3). Abbiamo trovato che la soluzione di incubazione e l&#39;influenza di tempo la capacità di DII per colorare sempre la cuticola (Tabella 1, Figura 4). Il metodo per DII neuroni colorazione dell&#39;ambiente esposto utilizza due ore di tempo di incubazione in M9 con Triton X-100 15. Un lavaggio iniziale di M9 con il tensioattivo Triton X-100 contribuisce a eliminare la contaminazione dalla superficie cuticolare e impedire agli animali di aggregazione. Tuttavia, incubando le tre ore gli animali in una soluzione colorante nel buffer stesso impedisce il colorante dalla colorazione della cuticola (Tabella 1).Questo può essere causato da lipidi in superficie nematode essere tolto dal trattamento più lungo nella soluzione detergente. L&#39;incubazione di animali in acqua con macchie Dii la cuticola, indicando che la soluzione salina M9 non è richiesto per la colorazione DII di cuticola. Anche se un protocollo a base di acqua è consigliato per neuronale colorazione DII 15, troviamo che gli animali trattati in questo modo appaiono spesso malsana. Lavaggio animali brevemente nello 0,5% Triton X-100 a M9, ​​seguito da tre ore di incubazione nella soluzione colorante a base di M9, fornisce colorazione coerente di strutture cuticolare e mantiene il benessere degli animali. Gli animali possono essere salvati dopo l&#39;osservazione e la manutenzione, permettendo analisi diretta a valle. Questo metodo è un comodo strumento che può essere utilizzato in molti studi, tra cui, ma non limitato a, la secrezione cuticolare e organizzazione, lo sviluppo delle cellule epidermiche, percorsi gene eterocronici, e l&#39;evoluzione dei nematodi.

<table border="1"><tbody><tr><td> Reagenti </td><td> Sinonimi </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Dii </td><td> 1,1 &#39;-Dioctadecyl-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine perclorato </td><td> Biotium, Inc. </td><td> 60010 </td><td> Magazzino di diluizione: 20 mg / mL in DMF diluizione di lavoro: 30 mg / mL </td></tr><tr><td> DMF </td><td> Dimetilformammide </td><td> Sigma-Aldrich, Inc. </td><td> D4551 </td><td></td></tr><tr><td> Tritone X-100 </td><td> Octylphenoxypolyethoxyethanol </td><td> VWR International, LLC. </td><td> EM-9410 </td><td></td></tr><tr><td> M9 </td><td> 22mm KH 2 PO 4, 42mm Na 2 HPO 4, 86 mm di NaCl, 1 mM MgSO 4 </td><td></td><td></td><td></td></tr><tr><td> NGM &lt;/ Td&gt; <td> Nematode crescita medio </td><td> IPM Scientific, Inc. </td><td> 11006-501 </td><td> Può essere preparato seguendo il protocollo NGM agar 18 </td></tr><tr><td> Agar-agar </td><td></td><td> EMD Chemicals, Inc. </td><td> 1,01614 </td><td> 4% in acqua </td></tr><tr><td> Levamisolo </td><td> Levamisolo cloridrato </td><td> Sigma-Aldrich, Inc. </td><td> 31742 </td><td> 100 mM - 1 levamisolo mM come richiesto </td></tr><tr><td> Microscopio diapositive </td><td></td><td> VWR International, LLC. </td><td> 16005-106 </td><td></td></tr><tr><td> Microscopio copertura occhiali </td><td></td><td> VWR International, LLC. </td><td> 16004-302 </td><td></td></tr><tr><td> Composto portata </td><td></td><td> Carl Zeiss, Inc. </td><td> A1M </td><td> Utilizzare gli obiettivi per soddisfare le esigenze della sperimentazione </td></tr><tr><td> TRITC o altri filtro compatibile </td><td> &amp;nbsp; </td><td> Chroma Technology Corp. </td><td> 49005 </td><td> ET - DsRed (TRITC/Cy3) atomizzate set di filtri </td></tr></tbody></table>

visualization of caenorhabditis elegans cuticular structures using the lipophilic vital dye dii

La cuticola di C. elegans è una struttura molto resistente che circonda l&#39;esterno dell&#39;animale 1-4. La cuticola non solo protegge l&#39;animale dall&#39;ambiente, ma determina anche la forma del corpo e svolge un ruolo nella motilità 4-6. Diversi strati secreto dalle cellule epidermiche comprendono la cuticola, tra cui una strato lipidico esterno 7. Creste circonferenziale nella cuticola chiamato modello annuli la lunghezza dell&#39;animale e sono presenti in tutte le fasi di sviluppo 8. Ali sono creste longitudinali che sono presenti durante le fasi specifiche di sviluppo, tra cui L1, dauer, e le fasi adulti 2,9. Le mutazioni nei geni che influenzano l&#39;organizzazione cuticolare collagene possono alterare la struttura cuticolare e morfologia del corpo animale 5,6,10,11. Mentre l&#39;imaging cuticolare usando la microscopia composto con ottica DIC è possibile, gli attuali metodi che mettono in risalto le strutture cuticolare comprendono fluorescenticento transgene espressione 12, colorazione anticorpale 13, e la microscopia elettronica 1. Etichettati germe di grano agglutinina (WGA) è stato utilizzato anche per visualizzare glicoproteine ​​cuticolare, ma è limitato nella risoluzione dei più fini strutture cuticolare 14. La colorazione della superficie cuticolare con colorante fluorescente è stato osservato, ma mai caratterizzato in dettaglio 15. Vi presentiamo un metodo per visualizzare in vivo cuticola C. elegans con il colorante rosso fluorescente lipofile DII (1,1 &#39;-dioctadecyl-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine perclorato), che è comunemente usato in C. elegans per visualizzare l&#39;ambiente neuroni esposti. Questo protocollo ottimizzato per la colorazione DII è un metodo semplice e robusto per la visualizzazione ad alta risoluzione fluorescenti di annuli, alae, vulva, coda di sesso maschile e ermafrodita coda picco di C. elegans.

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Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Vi presentiamo un metodo per visualizzare in vivo cuticola<em&gt; C. elegans</em&gt; Con il colorante rosso fluorescente lipofile DII (1,1 &#39;-dioctadecyl-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine perclorato), che viene comunemente usato in<em&gt; C. elegans</em&gt; Per visualizzare l&#39;ambiente neuroni esposti. Con questo protocollo ottimizzato, le strutture e le ali anulare cuticolare sono macchiati da DII e osservate al microscopio composto.

Visualizzazione di Caenorhabditis Elegans Strutture cuticola Utilizzando il Dye lipofili Vital, Dii

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

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Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

17.0K Views.  Texas A&M University System Health Science Center, College of Medicine. Vi presentiamo un metodo per visualizzare in vivo cuticola C. elegans Con il colorante rosso fluorescente lipofile DII (1,1 '-dioctadecyl-3, 3,3', 3'-tetramethylindocarbocyanine perclorato), che viene comunemente usato in C. elegans Per visualizzare l'ambiente neuroni esposti. Con questo protocollo ottimizzato, le strutture e le ali anulare cuticolare sono macchiati da DII e osservate al microscopio composto.

Video: Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Misura Caenorhabditis elegans Life Span in 96 piastre per microtitolazione Bene

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

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Biologia

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 ...

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 coltivazione di<em&gt; Caenorhabditis elegans</em&gt; In tre dimensioni in laboratorio

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 ...

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl&#8217;s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

DiI Perfusion come metodo per la visualizzazione vascolare in<em&gt; Ambystoma mexicanum</em

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Approcci quantitativi per studiare le strutture cellulari e la morfologia delle orgine in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

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.

Purificazione e analisi di 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 ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Utilizzo di Caenorhabditis elegans per lo screening delle interazioni chaperone tessuto-specifiche

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

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 ...

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based applications.

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Presented here is a description of the straightforward and relatively rapid isolation of Caenorhabditis elegans genomic DNA from one or a few animals using a commercially available tissue kit. The resulting gDNA preparation is a suitable template for PCR.

Estrazione su piccola scala di Caenorhabditis elegans DNA genomico

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and ...