Questo lavoro è stato sostenuto da un nuovo investigatore di Grant [2014R1A1A1005553] dalla Fondazione Nazionale delle Ricerche di Corea (NRF) per JIL; e Università di Yonsei Future Leader sovvenzione sfida [2015-22-0133] per JIL

1. Preparare Soluzioni per NGT-3D e NGB-3D <ol><li> Preparare le seguenti soluzioni sterili: 1 L di soluzione 0,1454 M di NaCl, 1 L di 1 M CaCl 2, 1 L di 1 M MgSO 4, lisogenia Broth (LB), 1 L di 1 M KPO 4 tampone (108,3 g di KH 2 PO 4 e 35,6 g di K 2 HPO 4, e riempire H 2 O a 1 L). Produzione di NGM utilizzando queste soluzioni può essere trovato in un precedente protocollo 10. </li><li> Le soluzioni autoclave a 121 ° C, 15 min. </li><li> Preparare 50 ml di 5 mg / ml soluzione colesterolo sterile. In una provetta conica da 50 ml, mescolare 0,25 g di colesterolo e 50 ml di etanolo 99,99% e mescolare bene. Non sterilizzare in autoclave. Sterilizzare la soluzione di colesterolo utilizzando una siringa da 50 ml con un filtro a siringa da 0,45 micron. Questo sarà anche eliminare ogni colesterolo non disciolto. </li><li> (Opzionale) Preparare 10 ml di una 150 mm di 2&#39;-desossi-5-fluorouridina (FUDR) stock. In un tubo conico da 15 ml, mescolare 0,3693 gdi FUDR e 10 ml di acqua distillata. Agitare bene. </li></ol> 2. Preparare Coltura dei batteri per NGT-3D e NGB-3D <ol><li> Inoculare 10 ml di brodo LB con i batteri. I batteri standard utilizzati per la C. elegans alimentazione è ceppo di E. coli OP50. </li><li> Cultura l&#39;inoculazione batterica a 37 ° C in un incubatore agitazione durante la notte. </li><li> In preparazione per una diluizione seriale, aliquote 9 ml di soluzione NaCl in sterili 15 ml provette coniche. Ad esempio, aliquota 9 ml in un totale di 7 tubi per fare una diluizione 10 -7 del ceppo OP50 E. coli. </li><li> Usando una pipetta sterile, 1.000 ml, pipetta 1 ml di coltura batterica o coltura batterica diluito nella nuova provetta sterile con 9 ml di soluzione di NaCl e vortice bene il composto 10 ml. Ripetere fino a raggiungere la diluizione batterica desiderato. NOTA: La diluizione batterica desiderata dipende dal tipo e dalle condizioni di batteri e le condizioni sperimentali esatti.Ad esempio, a 10 -6 - diluizione del ceppo OP50 E. coli 10 -8 era sufficiente a produrre da 1 - 200 colonie batteriche per 6,5 ml tubo NGT-3D. Worms affidabile sviluppati e riprodotti normalmente quando il numero delle colonie era finita 60, avvenuta ad una diluizione di 10 -6 - 10 -7. Per NGB-3D, usare una diluizione di 10 -8. </li></ol> 3. Fare NGT-3D e NGB-3D (200 ml) <ol><li> Dopo preparazione della coltura batterica, miscela 0,6 g di NaCl, 1 g granulato agar, e 0,5 g di peptone in un pallone da 500 ml. Inserire una ancoretta magnetica nel pallone. </li><li> Aggiungere 195 ml di acqua distillata e coprire la bocca del pallone con un foglio di alluminio. </li><li> Autoclave 121 ° C per 15 min. </li><li> Porre il pallone in autoclave a caldo su un piatto mescolare, e mescolare a velocità moderata per almeno 2 ore. Raffreddare il pallone a 40 ° C. Assicurati di raffreddare sufficientemente come si continua da qui a temperature elevate può portare a opacità del agar finito.Tuttavia, temperature più basse possono provocare il rapido indurimento di agar. <ol><li> (Facoltativo) Per accelerare il processo di raffreddamento, mettere il pallone in un 40 ° C bagnomaria 15 minuti prima di mettere su un piatto mescolare. </li></ol></li><li> Quando la temperatura del supporto agar raggiunge 40 ° C, aggiungere 200 ml 1 M CaCl 2, 200 ml di 5 mg / soluzione colesterolo ml, 200 ml 1 M MgSO 4 e 5 ml 1 M KPO 4 buffer come la soluzione continua a mescolare a concentrazioni finali di 1 mM CaCl 2, 5 ug / ml di colesterolo, 1 mM MgSO 4, 1 mM KPO 4. <ol><li> (Opzionale) per test di durata della vita NGT-3D aggiungere 80 ml di 150 mm FUDR ad una concentrazione finale di 120 micron 12. </li></ol></li><li> Aggiungere 6 ml di 10 ml diluiti coltura batterica dal punto 2.4 nel pallone direttamente. <ol><li> (Opzionale) per NGT-3D, rimuovere le 6 ml di terreni di coltura prima del punto 3.6 e tenere in caldo separatamente. Questo supporto sarà utilizzato per i batteri senzastrato superiore. </li></ol></li><li> Utilizzando le procedure sterili, erogare i media in una camera di cultura sterile. Assicurarsi che il coperchio della camera si chiude ermeticamente. <ol><li> (Opzionale) Per evitare colonie batteriche formazione sulla superficie superiore della agar, fare uno strato di priva di batteri agar dal punto 3.6.1 sulla sommità prima che il supporto dal punto 3.7 completamente indurisce. Ciò creerà una zona libera da batteri nella parte superiore della NGT-3D. </li><li> Per NGT-3D, versare 6,5 ml supporti nelle provette di plastica trasparenti 8 ml di fare uno strato di batteri agar, e dispensare accuratamente 200 ml di mezzi di comunicazione priva di batteri sulla parte superiore del supporto semi-indurito 3D per fare un sottile bacteria- strato libero sulla parte superiore. </li><li> Per NGB-3D, versare 65 ml di media nel 25 centimetri bottiglia coltura cellulare 2 plastica trasparente. Tale importo deve riempire il corpo della bottiglia 25 centimetri cultura 2 cellule. </li></ol></li><li> Lasciare camere verticalmente a temperatura ambiente per una settimana per consentire colonie batteriche di crescerenotevoli dimensioni del diametro minimo di 1 mm. <ol><li> (Opzionale) per test di durata della vita in NGT-3D, camere di coprire con un foglio di alluminio per evitare il degrado luce di FUDR. </li></ol></li></ol> 4. Misurare Fitness di Worm Popolazione al NGT-3D (Brood dimensione relativa del saggio) <ol><li> Scegliere un verme L4 fase utilizzando un filo di platino raccogliere e trasferire su un piatto NGM senza batteri. Lasciare verme di spostarsi liberamente per alcuni minuti per rimuovere batteri attaccati al suo corpo. </li><li> Ripetere il passaggio 4.1 e fare in modo che il worm è esente da batteri. In generale, due repliche di 4.1 sono sufficienti. </li><li> posizionare con cura il verme pulita sulla superficie del supporto 3D con un pick filo di platino. Il worm dovrebbe finalmente entrare l&#39;agar agar nella matrice 3D. </li><li> Chiudere il coperchio senza bloccare consentendo una certa aria nel tubo, ma impedendo l&#39;essiccazione dei media agar. </li><li> Incubare per 96 ore a 20 ° C. </li><li> Dopo 4 giorni, chiudere ermeticamente i coperchi e posizionare ilNGT-3D camera di cultura in un bagno d&#39;acqua C 88 ° per sciogliere l&#39;agar. Il calore uccide i vermi, ma i loro corpi rimangono intatti. NOTA: L&#39;utilizzo 8 ml tubi per NGT-3D, 20 - 30 minuti di incubazione è di solito sufficiente. </li><li> Usando una pipetta di vetro, trasferire i mezzi fuso su un piatto di plastica 9 centimetri di Petri. pipette di plastica non sono invitati, come i vermi possono spesso bastone a loro. </li><li> Utilizzando un microscopio stereoscopico dissezione trasmissione, contare il numero di vermi al L3, L4 e stadi adulti. Non contare i vermi che sono L2 fase e più giovani, come le generazioni F1 e F2 possono essere confusi qui. Così, questo test è un test relativo dimensione covata piuttosto che un dosaggio totale dimensioni covata. </li></ol> 5. Immagine e Record Worm Comportamento NGB-3D <ol><li> Scegli uno stadio verme L4 utilizzando un filo di platino raccogliere e rimuovere i batteri attaccati alla superficie trasferendo ad una piastra NGM senza batteri e permettendo di muoversi liberamente per alcuni minuti. </li><li> Ripetere il passaggio 5.1 per assicurarsi che il worm è esente da batteri. </li><li> posizionare accuratamente il verme pulito sul centro della superficie agar del NGB-3D vicino al collo della bottiglia con un pick filo di platino. Il worm dovrebbe finalmente entrare l&#39;agar agar nella matrice 3D. </li><li> Chiudere il coperchio senza bloccare consentendo una certa aria nel tubo, evitando l&#39;asciugatura dei media agar. </li><li> Immagine o registrare il verme sotto una dissezione stereo microscopio di trasmissione. Regolare la messa a fuoco come il verme si muove attraverso la matrice 3D. </li></ol>

<ol>
	<li>Corsi, A. K., Wightman, B., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Transparent+Window+into+Biology:+A+Primer+on+Caenorhabditis+elegans.">A Transparent Window into Biology: A Primer on Caenorhabditis elegans.</a> Genetics. 200 (2), 387-407 (2015).</li><li>Consortium, C. E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+sequence+of+the+nematode+C.+elegans:+a+platform+for+investigating+biology.">Genome sequence of the nematode C. elegans: a platform for investigating biology.</a> Science. 282 (5396), 2012-2018 (1998).</li><li>Choi, J. I., Yoon, K. H., Kalichamy, S. S., Yoon, S. S., Lee, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+natural+odor+attraction+between+lactic+acid+bacteria+and+the+nematode+Caenorhabditis+elegans.">A natural odor attraction between lactic acid bacteria and the nematode Caenorhabditis elegans.</a> ISME J. 10 (3), 558-567 (2016).</li><li>Gaertner, B. E., Phillips, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+as+a+platform+for+molecular+quantitative+genetics+and+the+systems+biology+of+natural+variation.">Caenorhabditis elegans as a platform for molecular quantitative genetics and the systems biology of natural variation.</a> Genet Res (Camb). 92 (5-6), 331-348 (2010).</li><li>Cutter, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+evolution+in+the+wild.">Caenorhabditis evolution in the wild.</a> Bioessays. 37 (9), 983-995 (2015).</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 (1), 71-94 (1974).</li><li>Felix, M. A., Braendle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+natural+history+of+Caenorhabditis+elegans.">The natural history of Caenorhabditis elegans.</a> Curr Biol. 20 (58), R965-R969 (2010).</li><li>Barriere, A., Felix, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+local+genetic+diversity+and+low+outcrossing+rate+in+Caenorhabditis+elegans+natural+populations.">High local genetic diversity and low outcrossing rate in Caenorhabditis elegans natural populations.</a> Curr Biol. 15 (13), 1176-1184 (2005).</li><li>Lee, T. Y., Yoon, K. H., Lee, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGT-3D:+a+simple+nematode+cultivation+system+to+study+Caenorhabditis+elegans+biology+in+3D.">NGT-3D: a simple nematode cultivation system to study Caenorhabditis elegans biology in 3D.</a> Biol Open. 5 (4), 529-534 (2016).</li><li>Lewis, J. A., Flemming, J. T., Epstein, H. F., Shakes, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Culture+Methods.">Basic Culture Methods.</a> Caenorhabditis elegans.: Modern Biological Analysis of an Organism. 48, 4-27 (1995).</li><li>Choi, S. Y., Yoon, K. H., Lee, J. I., Mitchell, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Violacein:+Properties+and+Production+of+a+Versatile+Bacterial+Pigment.">Violacein: Properties and Production of a Versatile Bacterial Pigment.</a> Biomed Res Int. , 465056 (2015).</li><li>Solis, G. M., Petrascheck, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Caenorhabditis+elegans+life+span+in+96+well+microtiter+plates.">Measuring Caenorhabditis elegans life span in 96 well microtiter plates.</a> J Vis Exp. 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The authors have nothing to disclose.

La coltivazione laboratorio di C. elegans utilizzando le piastre di terreni di crescita nematode classici è stato fondamentale per le centinaia di importanti scoperte che la ricerca in C. elegans ha fornito. Qui, vi presentiamo i nuovi metodi per coltivare C. elegans in un ambiente che rifletta più accuratamente i loro naturale habitat tridimensionali. Sebbene altri metodi sono stati usati per osservare C. elegans in 3D 13, questo è il primo protocollo che permette la coltivazione di vermi in una matrice solida 3D. I due metodi qui illustrati, NGT-3D e NGB-3D, permettono agli scienziati di porre domande di idoneità, lo sviluppo e la crescita in coltura 3D, e anche di immagini e registrare i vermi in 3D. Anche se NGT-3D e NGB-3D hanno alcune differenze con le piastre standard 2D NGM, essi sono simili a questo metodo originale e regolata solo per aggiungere la coltivazione a vite senza fine in asse z. Così, troviamo che lo sviluppo, la fertilità e la durata della vita procedono normalmente nelle nostre condizioni. Ilobiettivo era quello di creare un protocollo che ogni laboratorio può utilizzare per le loro ricerche. Questi metodi sono molto semplici, facilmente riproducibile ad un costo molto basso, e permettono la regolazione di protocolli per adattarsi esperimenti specifici degli utenti. Anche se tutti i passaggi nel protocollo sono importanti, il passo fondamentale nel rendere NGT-3D e NGB-3D è la temperatura di raffreddamento del agar dopo la sterilizzazione in autoclave a passaggi 3.4 a 3.6 del protocollo. Se la temperatura si raffredda troppo basse (parecchi gradi al di sotto di 40 ° C), l&#39;agar inizierà a indurirsi e le soluzioni aggiunti non si mescolerà correttamente nel agar. Tuttavia, se il raffreddamento è insufficiente (parecchi gradi superiori a 40 ° C), i batteri aggiunti possono morire e la soluzione colesterolo aggiunto possono offuscare l&#39;agar agar rendendo l&#39;inutile per gli esperimenti. Inoltre, un&#39;area di cautela al passo 4.6. Durante la fusione l&#39;agar NGT-3D nel bagno d&#39;acqua, assicurarsi di chiudere i coperchi strettamente per aiutare nella fusione. Tuttavia, il calore può causaretappi per pericolosamente &quot;pop&quot; off e diventare un proiettile. Per motivi di sicurezza è preferibile coprire il bagno d&#39;acqua. Molte parti del protocollo sono abbastanza adattabili all&#39;utente. Per esempio, abbiamo utilizzato una chiara provetta di plastica per NGT-3D e 25 cm 2 chiaro bottiglia coltura cellulare (detiene un totale di 65 ml) per il NGB-3D. Altri tipi di tubi trasparenti o bottiglie disponibili per l&#39;utente dovrebbe anche funzionare. Inoltre, la crescita batterica è regolabile. Ceppo di E. coli OP50 richiede generalmente circa una settimana a crescere fino a almeno 1 mm, che è stato preferito per i nostri esperimenti. Nella nostra esperienza, un po &#39;più corti e anche molto più lunghi tempi di crescita per i batteri non sembrano influenzare la crescita worm o la fertilità. Regolazione della concentrazione di agar, tuttavia, influenzerà il movimento senza fine. Concentrazioni inferiori di agar determinano vermi mostrano un movimento nuoto simile, e concentrazioni più elevate possono inibire movimento complessivo. Un motivo di preoccupazione in NGB-3D è stato il problema di condensae l&#39;accumulo di acqua. Come la calda agar liquido indurisce, inevitabilmente si forma della condensa nel tempo, soprattutto in corrispondenza dell&#39;interfaccia della bottiglia e l&#39;agar. Questo può comportare problemi per il verme, che mostra comportamenti nuoto in liquidi 14 e la crescita di batteri, che possono diffondere nel flacone dal liquido. Prevenzione della condensa è difficile. Esistono due possibili rimedi per questo. Innanzitutto, facciamo in modo che la vite senza fine può facilmente inserire l&#39;agar ponendolo nel centro della superficie agar al collo della bottiglia. In secondo luogo, abbiamo volutamente mantenere la concentrazione di batteri basso in NGB-3D, diminuendo così la probabilità che una colonia cresce all&#39;interfaccia di liquido e agar. Un&#39;altra preoccupazione è la formazione immagine di vermi in NGB-3D. Per aumentare la chiarezza e la qualità delle immagini di vermi in 3D, colonie dovrebbe essere vicino alla superficie, ma rimangono completamente incorporato nel agar. Abbiamo cercato di far crescere colonie incorporati solo vicino al surface ponendo un sottile strato di agar batteri contenenti la superficie, e la carta residua era priva di batteri. Tuttavia, il problema non è stato completamente risolto da questo metodo. Invece, abbiamo deciso di produrre molte bottiglie NGB-3D in una sola volta, e utilizzare solo quelli che contengono colonie vicino alla superficie incorporati, aumentando così la possibilità che un worm si sposterà a una colonia vicino alla superficie. Una possibilità che non abbiamo esplorato sta cambiando i media solidi a una matrice diversa da quella agar granulato impieghiamo qui. Imaging vermi nella direzione dell&#39;asse Z può anche essere difficile. Facciamo questo alzando e abbassando la messa a fuoco grossolana manualmente, ma se un programma automatizzato di monitoraggio esiste per questo, può essere molto utile. La speranza è che le tecniche qui presentate per la coltivazione di C. elegans in 3D saranno ampiamente utilizzati dai biologi nematodi. Molte domande rimangono circa la biologia verme, tra cui l&#39;invecchiamento e comportamenti in 3D, e se ciò che gli scienziati osservano in2D è rilevante per il verme in 3D. In effetti, un precedente studio con NGT-3D ha mostrato l&#39;impatto sul fitness riproduttiva che la coltivazione 3D ha avuto su un mutante sensoriale che riproduce normalmente in condizioni 2D 9. Inoltre, uno studio ha dimostrato comportamenti di movimento alterati in 3D rispetto al 2D 15. Infine, questo sistema può essere usato per iniziare a rispondere a domande se certe condizioni, mutanti o manipolazioni, vantaggi idoneità per l&#39;animale nel suo ambiente nativo 3D.

The study of the nematode Caenorhabditis elegans in the laboratory has led to seminal discoveries in the field of biology over the last five decades1. C. elegans was the first multicellular organism to have its genome sequenced in 19982, and it has been invaluable in understanding the contributions of individual genes to the development, physiology, and behavior of a whole organism. Scientists now are looking to further understand how these genes may contribute to the survival and reproductive fitness of organisms in their natural environments, asking questions about ecology and evolution at the genetic level3-5. 
C. elegans once again can provide an excellent system to answer these questions. However, little is known about C. elegans biology in natural nematode habitats, and there are no current methods to simulate controlled natural conditions of C. elegans in the laboratory. In the lab, C. elegans is cultivated on the surface of agar plates seeded with E. coli bacteria6. In nature, however, C. elegans and related nematodes can be found sparsely inhabiting soils throughout the globe, but they are specifically found thriving in rotting fruits and vegetative matter7,8. These three-dimensional (3D) complex environments are quite different from the simple 2D environments to which worms are exposed to in the laboratory. 
To begin to answer questions about the biology of nematodes in a more natural 3D setting, we have designed a 3D habitat for laboratory cultivation of nematodes we called Nematode Growth Tube 3D or NGT-3D for short9. The goal was to design a 3D growth system that allows for comparable growth, development, and fertility to the standard 2D Nematode Growth Media (NGM) plates10. This system supports the growth of bacteria and nematodes over their entire life cycles in 3D, allows worms to move and behave freely in three dimensions, and is easy and inexpensive to manufacture and employ. 
In the current study, we provide a step-by-step method to manufacture NGT-3D and evaluate worm development and fertility. In addition to assessing worm fitness in 3D, we sought to image, video, and assess worm behavior and physiology in 3D cultivation. Thus, in addition to NGT-3D, we present here an alternate method called Nematode Growth Bottle 3D or NGB-3D, for the microscopic imaging of C. elegans during 3D cultivation. This will be especially important for the study of known behaviors identified in 2D, and the identification of novel behaviors unique to 3D cultivation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>LB broth, Miller (Luria-Bertani)</td>
			<td>Difco</td>
			<td>224620</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>DAEJUNG</td>
			<td>7548-4400</td>
			<td>58.44 MW</td>
		</tr>
		<tr>
			<td>Agar, Granulated</td>
			<td>Difco</td>
			<td>214530</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Bacto</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride, dihydrate</td>
			<td>Bio Basic</td>
			<td>CD0050</td>
			<td>2*H2O; 147.02 MW</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Bio Basic</td>
			<td>CD0122</td>
			<td>386.67 MW</td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>B&#38;J</td>
			<td>RP090-1</td>
			<td>99.99%; 46.07 MW</td>
		</tr>
		<tr>
			<td>Magnesium sulfate, anhydrous</td>
			<td>Bio Basic</td>
			<td>MN1988</td>
			<td>120.37 MW</td>
		</tr>
		<tr>
			<td>Potassium phosphate, monobasic, anhydrous</td>
			<td>Bio Basic</td>
			<td>PB0445</td>
			<td>136.09 MW</td>
		</tr>
		<tr>
			<td>2&#39;-Deoxy-5-fluorouridine</td>
			<td>Tokyo Chemical Industry</td>
			<td>D2235</td>
			<td>246.19 MW</td>
		</tr>
		<tr>
			<td>Potassium phosphate, dibasic, anhydrous</td>
			<td>Bio Basic</td>
			<td>PB0447</td>
			<td>174.18 MW</td>
		</tr>
		<tr>
			<td>Multi-Purpose Test Tubes</td>
			<td>Stockwell Scientific</td>
			<td>ST.8570</td>
			<td>8 mL</td>
		</tr>
		<tr>
			<td>Test Tube Closures</td>
			<td>Stockwell Scientific</td>
			<td>ST.8575</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Flask</td>
			<td>SPL Lifescience</td>
			<td>70125</td>
			<td>25 cm^2</td>
		</tr>
		<tr>
			<td>Research Stereo Microscope</td>
			<td>Nikon</td>
			<td>SMZ18</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Definition Color Camera Head</td>
			<td>Nikon</td>
			<td>DS-Fi2</td>
			<td></td>
		</tr>
		<tr>
			<td>PC-Based Control Unit</td>
			<td>Nikon</td>
			<td>DS-U3</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements Basic Research, Microscope Imaging Software</td>
			<td>Nikon</td>
			<td>MQS32000</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

La costruzione di NGT-3D è un protocollo semplice e lineare che si traduce in una provetta agar-riempita con piccole colonie batteriche distanziati tutto il agar (Figura 1A). Worms possono muoversi liberamente attraverso la matrice agar, trovare e consumare le colonie batteriche. Per confermare se C. elegans possono riprodursi e di crescere normalmente in NGT-3D, abbiamo confrontato la fertilità e lo sviluppo larvale in 3D con piastre standard 2D NGM. Nel saggio dimensioni nidiata relativa, adulto C. elegans ermafroditi in NGT-3D riproducono bene come ermafroditi nelle piastre standard 2D NGM quando colonie batteriche sono abbondanti con almeno 60 colonie (Figura 1B). Inoltre, lo sviluppo larvale in NGT-3D procede anche normalmente quando ci sono un sacco di colonie batteriche con la maggioranza dei vermi nello stato adulto dopo 96 ore (Figura 1C). Tuttavia, se le colonie batteriche sono scarsinella matrice 3D, sia per la dimensione relativa covata (Figura 1B, barra a sinistra) e lo sviluppo larvale (Figura 1C, barra di sinistra) sono negativamente influenzati. Per verificare se l&#39;abitazione in 3D ha alcun effetto sulla fisiologia a lungo termine del worm, abbiamo condotto un test di durata a confronto piatti NGT-3D e NGM. La vita media per i vermi su NGT-3D è stato di 15,6 ± 3,6 giorni, rispetto a 14,8 ± 3,1 giorni per piastre standard NGM. Le curve di durata della vita per i vermi che vivono sul NGT-3D e NGM erano quasi identici. Quindi, sembra che i vermi sopravvivere ugualmente bene in condizioni 3D e 2D. Fabbricazione Il NGB-3D non è anche difficile e dovrebbe tradursi in un chiaro, bottiglia cultura agar-riempita come quello visto nella figura 2A. Per visualizzare facilmente le colonie batteriche, abbiamo espresso deoxyviolacein, un viola scuro metabolita batterica 11, nella OP50ceppo di E. coli (figure 2A, 2B; ceppo a richiesta). Vermi vivi possono essere esposte in una trasmissione stereo microscopio-dissezione (Figura 2B, supplementare Film 1). Stoccaggio di NGT-3D e NGB-3D a temperatura ambiente è sufficiente a mantenere entrambe le camere di coltura per un massimo di 1 mese. Essiccazione del agar non è una preoccupazione sia per NGT-3D o NGB-3D se viene applicato un plug-tipo o tappo a vite che si inserisce il tubo o bottiglia. <img alt="Figura 1" src="/files/ftp_upload/55048/55048fig1.jpg" /> Figura 1: Sviluppo, la fertilità e la durata della vita di C. elegans coltivate in NGT-3D è paragonabile a quella della NGM standard di 2D. (A) Immagini di NGT-3D in diverse diluizioni di batteri. a sinistra econtiene proprio una diluizione 5 x 10 -7, e la metà è una diluizione 1 x 10 -7. (B) Dimensione relativa nidiata di wild-type vermi in NGT-3D a meno di 60 colonie OP50 (n = 24), maggiore o uguale a 60 colonie (n = 14), o su piastre 2D NGM (n = 29). Le barre di errore indicano l&#39;errore standard. (C) Percentuale di vermi F1 generazione in L3, L4 o adulto stadio di sviluppo in NGT-3D con meno di 60 colonie OP50, maggiori o uguali a 60 colonie, o su piastre NGM 2D. Curva (D) Sopravvivenza di wild-type C. elegans in piastre NGT-3D o NGM. NS non indica significativamente diversa calcolato log-rank test. Questa cifra è stata modificata da Lee 9. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55048/55048fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src = &quot;/ files / ftp_upload / 55048 / 55048fig2.jpg&quot; /&gt; Figura 2: Imaging C. elegans in 3D utilizzando NGB-3D. (A, B) Immagini di NGB-3D; (C) Immagini di adulti C. elegans vicino a una colonia di ceppo OP50 E. coli che esprimono la viola pigmento deoxyviolacein scuro. barra della scala è di 500 micron. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55048/55048fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/55048/55048supmov.jpg" /> Film supplementare 1: Adulto C. elegans si avvicinano un E. coli OP50-violacé in colonia NGB-3D. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55048/Jove_Mov_1.mp4">Clicca qui per scaricare il file.</a>

Watch this Scientific Journal Video about Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory at JoVE.com

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

Vi presentiamo un metodo semplice per la costruzione di sistemi di coltivazione nematode 3D chiamati NGT-3D e NGB-3D. Questi possono essere utilizzati per studiare nematode fitness e comportamenti in habitat che sono più simili a naturali Caenorhabditis elegans habitat rispetto alle laboratorio C. elegans piastre di coltura 2D standard.

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 ...

cultivation-caenorhabditis-elegans-three-dimensions

Nanyang Technological University

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JoVE Journal

Biology

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

17.7K Views.  Yonsei University.  Vi presentiamo un metodo semplice per la costruzione di sistemi di coltivazione nematode 3D chiamati NGT-3D e NGB-3D. Questi possono essere utilizzati per studiare nematode fitness e comportamenti in habitat che sono più simili a naturali Caenorhabditis elegans habitat rispetto alle laboratorio C. elegans piastre di coltura 2D standard. 

Video: Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

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 ...

Ricerca

Biologia

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 ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

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 range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

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 ...

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, ...

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 ...

Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions

The voice of humans and most non-human mammals is generated in the larynx through self-sustaining oscillation of the vocal folds. Direct visual documentation of vocal fold vibration is challenging, particularly in non-human mammals. As an alternative, excised larynx experiments provide the opportunity to investigate vocal fold vibration under controlled physiological and physical conditions. However, the use of a full larynx merely provides a top view of the vocal folds, excluding crucial portions of the oscillating structures from observation during their interaction with aerodynamic forces. This limitation can be overcome by utilizing a hemi-larynx setup where one half of the larynx is mid-sagittally removed, providing both a superior and a lateral view of the remaining vocal fold during self-sustained oscillation.
Here, a step-by-step guide for the anatomical preparation of hemi-laryngeal structures and their mounting on the laboratory bench is given. Exemplary phonation of the hemi-larynx preparation is documented with high-speed video data captured by two synchronized cameras (superior and lateral views), showing three-dimensional vocal fold motion and corresponding time-varying contact area. The documentation of the hemi-larynx setup in this publication will facilitate application and reliable repeatability in experimental research, providing voice scientists with the potential to better understand the biomechanics of voice production.

This paper introduces a protocol for the preparation of hemi-larynx specimens facilitating a multi-dimensional view of vocal fold vibration, in order to investigate various biophysical aspects of voice production in humans and non-human mammals.

Hemi-laringeo Setup per lo studio delle vibrazioni del popolare vocale in tre dimensioni

The voice of humans and most non-human mammals is generated in the larynx through self-sustaining oscillation of the vocal folds. Direct visual ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

Un protocollo per laboratorio Housing di turchese Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

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 ...