Questo lavoro è stato sostenuto dal National Institutes of HealthGrants DK85035 e DK074797 (IH).

1. Ceppi Worm <ol><li> Ottenere C. elegans tipo selvatico ceppi Bristol N2 dal Caenorhabditis Genetics Center (CGC) (http://www.cbs.umn.edu/cgc) e li mantengono su piastre NGM seminato con E. coli ceppo OP50 7. Nota: verme transgenico ceppi IQ6011 (Phrg-1 :: GFP) utilizzati sono stati generati come descritto in precedenza 11. IQ6011 può essere richiesto all&#39;autore corrispondente. </li></ol> 2. Preparazione di Modified C. elegans Habitation e riproduzione Medium (mCeHR) Preparare mCeHR mezzi liquidi come descritto di seguito 12. Questo supporto liquido axenic permette ai vermi di crescere senza fonti di cibo supplementari. Eseguire tutte le manipolazioni di mezzi liquidi axeniche e worm axeniche utilizzando condizioni rigorosamente sterile come una cappa a flusso laminare. <ol><li> Ottenere il latte pastorizzato ultra senza grassi da un negozio di alimentari. Utilizzare il pro refrigeratodotto e non il prodotto temperatura ambiente. Prima di utilizzare in mCeHR mezzo, striscia il latte sul Brain Heart Infusion (BHI) piastre di agar ed incubare per 3 giorni a 30 ° C e 37 ° C per verificare la sterilità. Trasferire il latte a 50 ml provette sterili e conservare a -80 ° C </li><li> Preparare ciascuno dei seguenti componenti e combinare nell&#39;ordine e importi indicati per preparare 1 L di mCeHR (Tabella 1A): 10 ml di 2 mM colina diacido citrato, 10 ml di vitamina e fattore di crescita mix (vedi ricetta in Tabella 1B), 10 ml di 2,4 mM myo-inositolo, 10 ml di 2 mM cloruro emina, 250 ml di acqua deionizzata. Applicare l&#39;aspirazione e il filtro attraverso un gruppo filtro 0,22 micron. </li><li> Aggiungere 20 ml di miscela di acido nucleico (ricetta nella Tabella 1C), 100 ml di miscela di minerali (ricetta nella Tabella 1D), 20 ml di 170 mg / ml lattoalbumina idrolizzato, 20 ml di aminoacidi essenziali, 10 ml di aminoacidi non essenziali acidi, 20 ml di 450 mM KH 2 PO 4,50 ml di 1,45 M D-glucosio, 10 ml di 1 M HEPES, sale di sodio, 250 ml di acqua deionizzata. </li><li> Applicare aspirazione per filtrare sterilizzare i componenti. Rimuovere il filtro e aggiungere 1 ml di 5 mg / ml di colesterolo. Assicurarsi che il pH della media è di circa 6-6,5. Nota: questa soluzione può essere conservata a -80 ° C fino ad un anno. </li><li> Aggiungere 20% (v / v) ultra pastorizzato latte scremato organico al mezzo mCeHR prima dell&#39;uso. Usare una tecnica scrupolosamente sterile (ad esempio in una cappa a flusso laminare) all&#39;apertura e si dispensa il latte. Conservare il supporto a 4 ° C. </li></ol> 3. Preparare C. elegans per la Cultura in axeniche mCeHR Liquid Media <ol><li> Crescere vermi dieci 60 millimetri piastre NGM finché ci sono molti vermi gravide e una quantità minima di OP50 E. coli sulle piastre. </li><li> Rimuovere le viti senza fine da ciascuna piastra da risciacquo con 5 ml di tampone M9 (ricetta in Tabella 1F), e combinare in una provetta conica da 50 ml. </li><li> Lasciare i vermi a settle lasciando il tubo riposare per 5 a 10 minuti poi rimuovere con attenzione il surnatante contenente la E. coli. </li><li> Ripetere i punti 3.2 e 3.3 2x per rimuovere la maggior quantità di batteri residui più possibile prima di continuare la procedura. </li><li> Risospendere i vermi in 0,1 N di NaCl in un volume che è un multiplo di tre, per esempio 1,5 ml, 3 ml o 6 ml in cui i singoli vermi può essere visto nel tubo. </li><li> Candeggiare i vermi aggiungendo NaOH 5 N e 5% soluzione di ipoclorito di sodio (candeggina) in un rapporto di 01:02:06 alla sospensione verme. Per esempio, 1 ml di NaOH 5 N: 2 ml 5% di ipoclorito di sodio: 6 ml della sospensione verme. Mescolare il NaOH e candeggina prima di aggiungere alla sospensione verme. </li><li> Vortex la soluzione energicamente fino vermi gravide vengono sciolti e solo uova sono visibili all&#39;interno della sospensione (circa 5 a 10 min). Monitorare il processo utilizzando un microscopio a contrasto di fase con un obiettivo 10X. </li><li> Dopo che i vermi hanno completamente sciolto, pellet immediatamente l&#39;uova a 800 xg per 45 secondi a 4 ° C. Rimuovere il surnatante e lavare il pellet due volte con 10 ml di acqua sterile per centrifugazione il pellet a 800 xg per 45 sec a 4 ° C. </li><li> Dopo lo sbiancamento, trasferire il pellet uovo ad un tessuto beuta da 25 cm 2 di coltura contenente 10 ml di media mCeHR integrato con 100 pg / ml di tetraciclina. Effettuare questa procedura utilizzando tecniche sterili. Se lo si desidera, aggiungere ulteriori antibiotici, di cui 250 mg / ml di acido nalidixico, per prevenire la crescita batterica nelle colture liquide axeniche. </li><li> Incubare le colture liquide a 20 ° C in un incubatore agitazione a circa 70 rpm. </li><li> Controllare i vermi ogni giorno per notare la fase di sviluppo e il tasso di crescita. Nota: Worms inizialmente crescere lentamente e richiedono 7 a 10 giorni per diventare gravida. Tuttavia, come i vermi acclimatarsi al mezzo liquido il tempo di generazione diminuisce di circa 4 giorni per tipo selvatico (N2) vermi. </li><li> Dopo i vermi hanno svipato alla fase gravido in mezzi liquidi, trasferire la cultura verme in una provetta conica e agglomerare le vermi a 800 xg per 5 minuti a 4 ° C utilizzando un rotore oscillante. </li><li> Rimuovere il surnatante e delicatamente risospendere il pellet verme in un volume uguale di 0,1 M NaCl. Ad esempio, utilizzare 10 ml di 0,1 M NaCl per i vermi coltivate in 10 ml di mCeHR. Lasciare i vermi di stabilirsi per 5 minuti in ghiaccio. </li><li> Effettuare la procedura di sbiancamento come sopra descritto nei passaggi 3,5-3,8, e permettere ai vermi di crescere per una seconda generazione nei mezzi liquidi axeniche. Se necessario, aggiungere di nuovo l&#39;antibiotico per inibire la crescita batterica. </li><li> Anche in questo caso consentono ai vermi di diventare gravida poi pellet e risospendere i vermi in 0.1 M NaCl come descritto nei punti 3.12 e 3.13, poi candeggiare nuovo come descritto nei passaggi 3,5-3,8, per produrre una popolazione sincronizzato. Crescere i vermi larve L1 in mezzi liquidi senza antibiotici da ora in poi. </li></ol> 4. Sincronizzazione Worms da LiquidCultura <ol><li> Pellet e risospendere i vermi a 0,1 M NaCl come sopra descritto nei passaggi 3.12 e 3.13. Bleach come sopra descritto nei passaggi 3,5-3,8. </li><li> Risospendere il pellet uovo in 10 ml di tampone M9 e permettere alle larve di schiudono O / N a 20 ° C su una piattaforma agitatore rotante. Nota: Le uova si schiudono in larve L1, ma non si svilupperanno ulteriormente, sincronizzando i vermi nella fase L1. </li><li> Per mantenere e vermi sottocultura, pellet L1 larve a 800 xg per 5 minuti, poi risospendere le larve in 10 ml di mCeHR e il trasferimento ad un pallone 25 centimetri 2. Lasciare i vermi di crescere a 20 ° C su una piattaforma rotante. Mantenere una densità massima di 3.000 ml / worm / cm 2 per assicurare una nutrizione adeguata per i vermi. Nota: In questa fase, gli antibiotici non sono tenuti a mantenere condizioni axeniche quando le procedure siano effettuate con tecniche sterili in una cappa a flusso laminare. Worms devono essere controllate quotidianamente per monitorare la crescita. </li></ol> 5. Liberozing e scongelamento Worms per colture axeniche medie <ol><li> Congelare le culture verme asincroni axeniche o sincronizzato larve L1 in azoto liquido. Worm pellet a 800 xg per 5 minuti poi risospendere in tampone di S (6,45 KHPO mm 4, 43.55 KHPO mm 4, 146,25 mM NaCl) con 0,5 ml per ogni flacone. Aggiungere un volume uguale di tampone S con il 30% v / v glicerolo ad ogni flacone e trasferire i vermi a -80 ° C prima della conservazione a lungo termine in N 2 liquido. Congelare circa 1 x 10 6 vermi per ml in ogni fiala. </li><li> Scongelare vermi incubando le fiale a 37 ° C per circa 2 min fino a quasi tutto il ghiaccio è fuso. In una cappa a flusso laminare, trasferire i vermi in un pallone sterile contenente mCeHR e metterli a 20 ° C. </li></ol> 6. Determinare l&#39;effetto di Hemin concentrazione sulla crescita e la riproduzione in mCeHR <ol><li> Utilizzare supporti mCeHR axeniche per saggiare la crescita dipende nutrienti di C. elegans. Per determinare l&#39;effetto of emina concentrazione sulla crescita senza fine e lo sviluppo, l&#39;uso sincronizzato vermi N2 axeniche o di altri ceppi di interesse. Sincronizzare L1 larve come descritto sopra nella sezione 4. </li><li> Il giorno successivo, pellet e contare sincronizzato larve L1 e portare ad 1 verme per ogni ml di mezzi axeniche. Effettuare 10 mM cloruro emina in 0,3 M NH 4 OH e regolare a pH 8 mediante HCl concentrato. </li><li> Aggiungere 800 microlitri di media mCeHR ad una piastra da 24 pozzetti. Aggiungere 100 vermi per ciascun pozzetto in 100 microlitri di media. Aggiungere cloruro di emina a ciascun pozzetto in concentrazioni comprese tra 0 mM, 1,5 mM a 1.000 pM in triplice copia. Assicurarsi che tutti i pozzetti contengono la stessa concentrazione di 0,3 M NH 4 OH. Risospeso delicatamente i worm prima il pipettaggio per garantire che il 100 L1 larve sono aggiunti a ciascun pozzetto. </li><li> Esaminare i vermi ogni giorno e notare la crescita di ciascuna concentrazione in ciascun pozzetto. Contare il numero di vermi dopo 9 giorni di incubazione e tracciare il numero di worm / ml corrispondenti a ciascuna emeconcentrazione (Figura 1). </li></ol> 7. Effetto della Hemin concentrazione su eme Worms sensori <ol><li> Incubare sincronizzati L1 vermi sensore eme (Phrg-1 :: GFP) in mCeHR contenente 4 micron, 8 micron, 10 micron e 20 micron emina. </li><li> Immagine worm utilizzando la microscopia confocale a fluorescenza, 48 ore dopo incubazione (Figura 2). </li></ol> 8. Utilizzando mCeHR per generare transgenici C. elegans Utilizzo di microparticelle Bombardamento Nota: la procedura per la generazione e la realizzazione di microparticelle bombardamento con vermi coltivate in mCeHR unc-119 (ED3) è illustrato nella figura 3 13. <ol><li> Worm Preparazione <ol><li> Trasferire circa 1 x 10 6 asincroni unc-119 (ED3 vermi) in 90 ml di mezzi mCeHR in A 175 cm 2 matraccio una settimana prima del bombardamento. Lasciare i vermi di crescere a 20 ° C ona piattaforma di agitazione a ~ 70 rpm (Figura 3-1). Nota: Una settimana più tardi, la densità verme dovrebbe essere significativamente maggiore con molti adulti vermi gravide. Circa 3 x 10 7 vermi saranno necessari per microparticelle bombardamento. </li><li> Freddo JM109 E. coli testa di serie 10 piatti cm NGM sul ghiaccio. </li><li> Trasferire i vermi axeniche a due tubi da 50 ml coniche e centrifugare i vermi a 800 xg per 2 minuti. Aspirare il surnatante e sospendere nuovamente le viti senza fine in ciascun tubo in 50 ml di tampone M9 (Figura 3-2). </li><li> Lasciare i vermi adulti a pellet per gravità per 10-15 minuti sul ghiaccio. Nota: Il pellet 2 ml deve contenere ora&gt; 90% vermi adulti. Questo pellet 2 ml dovrebbe avere circa 5 x 10 6 vermi ed è sufficiente per un bombardamento microparticelle. </li><li> Rivestire l&#39;intera superficie del refrigerata JM109 seminato piastra NGM con il pellet 2 ml di vermi. Che il liquido sia completamente assorbito poi lasciare la piastraper 30 min in ghiaccio prima di proseguire (Figura 3-3). </li></ol></li><li> Preparazione di particelle d&#39;oro <ol><li> Pesare 30 mg di particelle d&#39;oro in una provetta siliconato. Aggiungere 1 ml di etanolo al 70% e agitare la provetta per 5 min. </li><li> Lasciare che il tubo a sedere per 15 minuti, poi centrifugare a 6.000 xg per 10 secondi per far sedimentare le particelle d&#39;oro. Rimuovere il surnatante utilizzando un puntale cautamente la punta verso il lato opposto del tubo di particelle d&#39;oro. </li><li> Lavare le particelle d&#39;oro con 1 ml di acqua sterile, vortex per 1 min e centrifugare brevemente la provetta a 6.000 xg per 10 sec. Ripetere la fase di lavaggio due volte, poi risospendere le particelle d&#39;oro in 0,5 ml di soluzione sterile 50% glicerolo. Nota: La concentrazione finale delle particelle d&#39;oro sarà di 60 mg / ml e può essere conservato per 1-2 mesi a 4 ° C. </li></ol></li><li> Preparare la miscela di particelle di DNA-oro <ol><li> Combinare 10 pg di DNA plasmidico desiderato con 10 &amp;# 956; g del unc-119 di salvataggio plasmide in un siliconato 1,5 ml provetta. Aggiungere 50 ml di acqua deionizzata e 100 ml di ben risospeso soluzione di particelle d&#39;oro poi vortex per 1 min. </li><li> Aggiungere 150 ml di 2,5 M CaCl 2 e agitare nuovamente la soluzione per 1 min. A questa soluzione, aggiungere 60 ml di 0,1 M spermidina e vortex per 3-5 min. </li><li> Pulse centrifugare la soluzione a 6.000 xg per 10 secondi, rimuovere il surnatante e aggiungere 300 ml di etanolo al 70%. Vortex bene per 1 min, impulso centrifugare a 6000 xg, rimuovere il supernatante e risospendere in 170 ml di etanolo al 100%. Vigorosamente agitare la soluzione per 5-10 minuti poi pulsano centrifugare e rimuovere il surnatante. </li></ol></li><li> Bombardamento (Figura 3-3) Nota: Questo protocollo è eseguita con il sistema di erogazione di particelle specificato nella tabella dei materiali / Attrezzatura. Seguire le istruzioni per lo strumento consegna particella disponibili. <ol> &lt;li&gt; Inserire un foglio di carta velina in un piatto di 15 centimetri. Usando le pinzette, immergere sette macrocarriers singolarmente in 100% di etanolo e stenderli sul tessuto ad asciugare. </li><li> Pulire accuratamente la camera di biolistic, titolare macrocarrier e piastra bersaglio con etanolo al 70%. Pulire e sterilizzare gli schermi di arresto prima di ogni procedura di bombardamento. </li><li> Caricare i macrocarrriers nel supporto utilizzando pinzette e premere nel supporto utilizzando lo strumento dei posti a sedere. </li><li> Distribuire uniformemente 20 ml del DNA rivestiti particelle d&#39;oro nel centro di ogni macrocarrier e poi lasciare che la soluzione si asciughi completamente. </li><li> Pulire le due parti del divisore pressione con etanolo e assemblare con etanolo puliti dischi di rottura. Avvitare il divisore a pressione montate e dischi di rottura nella camera di bombardamento. </li><li> Montare uno schermo arresto autoclave con il titolare macrocarrier e posizionare l&#39;apparecchio al scaffale di metallo richiesto e bloccare in posizione. </li><li> Inserire il MACR assemblataocarrier apparecchio nella camera biolistic nello slot assegnato sotto del divisore pressione e allinearsi con il divisore pressione. </li><li> Nastro il piatto NGM aperto con i vermi sul ripiano piastra bersaglio e chiudere la porta della camera. </li><li> Per bombardare worm, regolare la pressione a quella necessaria per rompere i dischi di rottura. Accendere il vuoto a 28 pollici di pressione Hg, quindi premere il pulsante di fuoco fino a quando i dischi di rottura. </li><li> Rilasciare il vuoto e rimuovere la piastra NGM dall&#39;apparecchio. Lavare i vermi dalla piastra e ridistribuire in venti 10 piatti cm NGM seminato con JM109 E. coli (Figura 3-4). </li><li> Lasciare i vermi di crescere per 10 a 14 giorni a 25 ° C e morire di fame i piatti. Worms salvati dal difetto unc-119 avranno il movimento normale e possono essere identificate in questo momento. Scegli almeno 10 &quot;di tipo selvatico&quot; vermi da lastre separate per ulteriori analisi in quanto questi rappresentano linee transgeniche indipendenti (Figure 3-5). </li></ol></li></ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologous+gene+targeting+in+Caenorhabditis+elegans+by+biolistic+transformation.">Homologous gene targeting in Caenorhabditis elegans by biolistic transformation.</a> Nucleic acids research. 32, e40 (2004).</li><li>Semple, J. I., Biondini, L., Lehner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+transgenic+nematodes+by+bombardment+and+antibiotic+selection.">Generating transgenic nematodes by bombardment and antibiotic selection.</a> Nature Methods. 9, 118-119 (2012).</li></ol>

Gli autori dichiarano non ci sono interessi economici in competizione o conflitto di interessi.

In questo protocollo vi presentiamo un axenic mCeHR mezzi liquidi modificato che consente una rapida C. generazione elegans con produzione di un gran numero di vermi. Questo supporto mostra diversi vantaggi come i vermi sono coltivate senza contaminare E. coli o sottoprodotti batterici e possono essere sfruttati in studi nutrizionali e tossicologici. L&#39;uso di E. coli o altri batteri in detti studi ha diversi inconvenienti. Ad esempio, la crescita dei batteri può cambiare in varie...

Il nematode del suolo, Caenorhabditis elegans, è un potente organismo modello utilizzato in numerosi studi dalla genetica alla tossicologia. Come risultato della sua dimensione 1 mm tempo di generazione rapida di quattro giorni facilità di coltivazione, e un gran numero di progenie, questi nematodi sono stati utilizzati in un numero di schermi genetici e farmacologici 1, 2. Ricercatori approfittare di questo worm per identificare molecole e percorsi conservati nei sistemi di vertebrati. Questi percorsi sono segnali di morte cellulare, percorsi di invecchiamento e del metabolismo, e il sistema nervoso 3-6. Inoltre, la trasparenza di C. elegans consente la generazione di linee transgeniche utilizzando reporter proteine ​​fluorescenti, che possono essere visualizzati direttamente per analizzare i modelli di espressione genica e localizzazione delle proteine. In molti studi questo nematode è coltivato su una superficie a base di agar-solido utilizzando nematode mezzo di crescita (NGM) piastre o in lculture iquid seminati con Escherichia coli come fonte di cibo 7,8. Queste fonti di cibo batteriche possono confondere studi biochimici e tossicologici con interferenza da batteri sottoprodotti che influenzano l&#39;interpretazione dei risultati. Per evitare questi effetti compounding, C. elegans possono essere coltivate in un mezzo liquido axeniche che è privo di batteri come fonte di cibo. Usando questo supporto, abbiamo coltivato con successo milioni di vermi altamente sincronizzati per molti di serie C. protocolli elegans, compresa l&#39;analisi microarray di geni differenzialmente regolati in C. elegans esposto a diverse concentrazioni eme, e produzione di vermi transgenici utilizzando gene bombardamento. Questo supporto è chimicamente definita e modificata da una ricetta originale formulata dal Dr. Eric Clegg 9. Usando questo supporto mCeHR, abbiamo identificato con successo geni coinvolti nella eme omeostasi, riferiti ai geni come eme responsivi (HRG s) 10, che avrebbenon sarebbe stato possibile in condizioni di crescita regolari che utilizzano piastre di agar NGM seminato con E. coli. In questo protocollo si descrive la procedura per l&#39;introduzione e il mantenimento C. elegans cresciuti su E. coli per la axenic mCeHR e utilizzare questo metodo per ottenere un gran numero di vermi per produrre transgenico C. Linee elegans utilizzando microparticelle bombardamento. Abbiamo presenti anche studi che dimostrano l&#39;utilità di utilizzare supporti axeniche per la determinazione del fabbisogno nutrizionale di C. elegans utilizzando eme come esempio. Questi studi mostrano che utilizzando mCeHR supporti consente la rapida crescita di un gran numero di C. elegans per molte applicazioni a valle utilizzate dai ricercatori worm.

<table border="0" cellpadding="0" cellspacing="0" width="754">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">MgCl2.6H2O</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">M-2393</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium citrate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">S-4641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium citrate.H2O</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-1722</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CuCl2.2H2O</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:121px;">C455-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MnCl2.4H2O</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:121px;">M87-100</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">ZnCl2</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">Z-0152</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Fe(NH4)2(SO4)2.6H2O</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">F-1018</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CaCl2.2H2O</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:121px;">C70-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine 5 -monophosphate, sodium salt</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">A-1752</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytidine 5 -phosphate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">C-1006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanosine 2 - and3&#160; -monophosphate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">G-8002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uridine 5 -phosphate, disodium salt</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">U-6375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thymine&#160;</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">T0376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">N-Acetylglucosamine</td>
			<td style="width:108px;">Sigma</td>
			<td>A3286</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">DL-Alanine</td>
			<td style="width:108px;">Fisher</td>
			<td>S25648&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">p-Aminobenzoic Acid</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">A-9878</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Biotin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">B-4639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Cyanocobalamine (B-12)</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">V-2876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Folinate (Ca)&#160;</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">F-7878</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Niacin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">N-0761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Niacinamide</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">N-3376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pantetheine</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-2125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pantothenate (Ca)</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-6292</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pteroylglutamic Acid (Folic Acid)</td>
			<td style="width:108px;">ACRCS</td>
			<td style="width:121px;">21663-0100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pyridoxal 5&#39;-phosphate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-3657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pyridoxamine.2HCl</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-9158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pyridoxine.HCl</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-6280</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">Riboflavin 5-PO4(Na)</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">R-7774</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Thiamine.HCl</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">T-1270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">DL-6,8-Thioctic Acid</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">T-1395</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">KH2PO4</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-5379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Choline di-acid citrate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">C-2004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">myo-Inositol</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">I-5125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">D-Glucose</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">G-7520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lactalbumin enzymatic hydrolysate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">L-9010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brain Heart Infusion</td>
			<td style="width:108px;">BD</td>
			<td style="width:121px;">211065</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Hemin chloride</td>
			<td style="width:108px;">Frontier Scientific</td>
			<td style="width:121px;">H651-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">HEPES, Na salt</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">H-3784</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Cholesterol</td>
			<td style="width:108px;">J.T. Baker</td>
			<td style="width:121px;">F676-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids</td>
			<td style="width:108px;">Invitrogen</td>
			<td>11140-076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Amino Acids Solution</td>
			<td style="width:108px;">Invitrogen</td>
			<td>11130-051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nalidixic acid sodium salt</td>
			<td style="width:108px;">Sigma</td>
			<td>N4382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetracycline Hydrochloride</td>
			<td style="width:108px;">MP Biomedicals</td>
			<td>2194542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biolistic Delivery System</td>
			<td style="width:108px;">BioRad</td>
			<td>165-2257</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Gold particles (Au Powder)&#160;</td>
			<td style="width:108px;">&#160;Ferro Electronic Material Systems</td>
			<td style="width:121px;">6420 2504, JZP01010KM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">or</td>
			<td style="width:108px;"></td>
			<td style="width:121px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gold Particles 1.0 &#956;m</td>
			<td style="width:108px;">BioRad</td>
			<td>&#160;165-2263</td>
			<td></td>
		</tr>
	</tbody>
</table>

culturing caenorhabditis elegans in axenic liquid media and creation of transgenic worms by microparticle bombardment

In questo protocollo, presentiamo i materiali necessari, e la procedura per fare C modificati. Legans e assuefazione e della Riproduzione media (mCeHR). Inoltre, la procedura per l&#39;esposizione e acclimatazione C. elegans cresciuti su E. coli a axeniche mezzi liquidi sono descritti. Infine, gli esperimenti a valle che utilizzano axenic C. elegans illustrano i benefici di questa procedura. La capacità di analizzare e determinare C. fabbisogno nutritivo elegans è stato illustrato da una crescente N2 wild type vermi in mezzi liquidi axeniche con concentrazioni variabili eme. Questa procedura può essere replicata con altri nutrienti per determinare la concentrazione ottimale per la crescita e lo sviluppo verme o, per determinare gli effetti tossicologici dei trattamenti farmacologici. Gli effetti di varie concentrazioni eme sulla crescita di tipo selvaggio vermi sono stati determinati mediante osservazione microscopica qualitativa e quantificazione tegli numero di worm che crescevano in ciascuna concentrazione eme. Inoltre, l&#39;effetto di varie concentrazioni di nutrienti può essere saggiata utilizzando vermi che esprimono sensori fluorescenti che rispondono alle variazioni del nutriente di interesse. Inoltre, un gran numero di vermi erano facilmente prodotti per la generazione di transgenici C. elegans utilizzando microparticelle bombardamento.

Coltura C. elegans in axeniche aiuti mezzo liquido nella determinazione di sostanze nutritive che sono richiesti dai vermi, senza interferenze da parte di metaboliti secondari prodotti da E. coli. Worm di tipo selvatico N2 acclimatare i media mCeHR entro tre generazioni e mostrano una crescita paragonabile a vermi cresciuti su piastre NGM batteriche. Infatti, questi vermi diventano gravide entro 4 giorni, rispetto a 3,5 giorni per i vermi coltivati ​​sui batteri OP50. U...

Watch this Scientific Journal Video about Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment at JoVE.com

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans è di solito coltivate su piastre di agar solidi o liquidi in colture seminate con E. coli. Per evitare sottoprodotti batterici da confondere studi tossicologici e nutrizionali, abbiamo utilizzato un mezzo liquido axenic, CeHR, crescere e sincronizzare un gran numero di vermi per una gamma di applicazioni a valle.

Culturing<em&gt; Caenorhabditis elegans</em &gt; In axeniche Liquid Media e la creazione di transgeniche Worms da microparticelle Bombardamento

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

culturing-caenorhabditis-elegans-axenic-liquid-media-creation

Research

JoVE Journal

Biology

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

16.9K Views.  University of Maryland.  C. elegans è di solito coltivate su piastre di agar solidi o liquidi in colture seminate con E. coli. Per evitare sottoprodotti batterici da confondere studi tossicologici e nutrizionali, abbiamo utilizzato un mezzo liquido axenic, CeHR, crescere e sincronizzare un gran numero di vermi per una gamma di applicazioni a valle. 

Video: Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

Measuring Caenorhabditis elegans Life Span on Solid Media

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

Measuring Caenorhabditis elegans Life Span on Solid Media

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

Misura Caenorhabditis elegans Life Span su terreni solidi

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

Ricerca

Biologia

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

Generazione di transgenici C. elegans Di Trasformazione Biolistic

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

Isolamento e In vitro Attivazione di Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

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

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

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

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

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

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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

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

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

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

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

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

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

Prostaglandina estrazione e l&#39;analisi in<em&gt; Caenorhabditis elegans</em

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

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

Coltura di cellule umane nasale epiteliali presso l&#39;Air Interface Liquid

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

Un Screenable<em&gt; In Vivo</em&gt; Saggio per mitocondriale modulatori Uso Transgenic Bioluminescent<em&gt; Caenorhabditis elegans</em

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

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

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

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

Combinato di nucleotidi e proteina estrazioni in Caenorhabditis elegans

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

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 ...