Questo lavoro è stato supportato da una Glenn / AFAR scoperte nel Premio Gerontologia e NIH di Grant 1R01AG031108-01 a MKGS è supportato dal NIH formazione concedere P30AG013280. MK è una Ellison Medical Foundation Scholar Nuove invecchiamento.

Parte 1: Preparare nematode crescita media (NGM) piastre Questa sezione descrive la preparazione dei piatti NGM solido per l&#39;uso nella sperimentazione durata della vita. Un esperimento di base durata della vita richiede due tipi di piastre: piastre NGM standard, che non contengono additivi, e Amp / FUDR piatti, che hanno entrambi ampicillina (Amp) e fluorodeoxyuridine (FUDR) aggiunto al NGM. L&#39;ampicillina è usata per prevenire la contaminazione batterica stranieri. FUDR inibisce la divisione cellulare, riduce la produzione di uova, e previene le uova da cova. L&#39;uso di FUDR per l&#39;analisi longevità non influisce durata vita adulta ed elimina la necessità di trasferire i vermi ogni pochi giorni al fine di separarli dalle larve in crescita. Entrambi i tipi di piastre sono seminati con E. coli OP50 batteri, che viene successivamente ucciso da esposizione ai raggi UV. <ol><li> Preparare NGM (vedi sezione 5 per ricetta e le note di stoccaggio) e piastre Petri etichetta. Avrete bisogno di 1 60 mm Piastra di Petri per 10 ml di NGM. Se si inizia con NGM solido precedentemente preparato, passare al punto 1.2. Se si inizia con NGM appena sterilizzati in autoclave, passare al punto 1.4. </li><li> Completamente sciogliere NGM solido microonde in alto di 30 impulsi secondo. Mezzi di turbolenza tra gli impulsi per evitare bolle di gas pressurizzato da costruire. </li><li> Luogo NGM liquido in bagno d&#39;acqua a 55 ° C o in panchina a raffreddare. </li><li> Una volta NGM raggiunge i 55 ° C, aggiungere 33 ml di FUDR 150 mm e 100 ml di 100 mg / ml per 100 ml Ampicillina NGM (per Amp / FUDR piatti) e mescolare. Per piastre NGM senza farmaci aggiuntivi passare direttamente al punto 1.5. </li><li> Usando la tecnica sterile, pipetta 10 mL NGM in ogni piatto 60 millimetri di Petri. Evitare la formazione di bolle, se possibile. Se le bolle si formano, possono essere spuntato con un puntale. </li><li> Lasciare le piastre sul banco con coperchi a permettere NGM solidificare e asciugare. Se possibile, lasciare le piastre sul banco per 2 giorni prima di aggiungere i batteri, ma 1 giorno funzionerà se le targhe sono necessarie prima. </li><li> Il giorno prima le piastre finitura essiccazione, liquido cultura seme LB con una singola colonia da una vena fresca di E. coli OP50 batteri su agar LB. Si dovrebbe preparare almeno 1 ml di una notte OP50 cultura a 60 mm. </li><li> Luogo OP50 cultura in 37 ° C shaker e permettere ai batteri di crescere durante la notte (la cultura dovrebbe raggiungere la saturazione). </li><li> Pellet OP50 batteri facendo girare a 3.500 g per 10 minuti. </li><li> Rimuovere il 90% dei batteri surnatante e risospendere di concentrarsi cultura 10x. </li><li> Pipettare 100 l di 10x concentrata OP50 cultura al centro delle piastre NGM solidificato. Agitare delicatamente piatti di diffondere la cultura dei batteri, se necessario (idealmente batteri dovrebbero coprire l&#39;area centrale della NGM senza entrare vicino alle mura della piastra). Cercate di evitare di toccare la punta della pipetta sulla superficie della NGM, come difetti nella superficie del supporto permettono i vermi a scavare nel NGM. </li><li> Lasciare le piastre sul banco durante la notte per permettere ai batteri di cultura asciugare sul NGM solido (in genere dura circa 24 ore). </li><li> Una volta asciutto, esporre la superficie delle piastre ad una dose UV sufficiente ad arrestare la crescita dei batteri. Se si utilizza un Stratalinker UV Stratagene 2400: <ul><li> Piastre posto nella Stratalinker e togliere coperchi </li><li> Chiudere la porta e accendere la Stratalinker </li><li> &#39;Energia&#39; stampa </li><li> Inserisci &#39;9999 &#39;usando la tastiera </li><li> &#39;Start&#39; stampa </li><li> Le piastre saranno esposti a raggi UV per circa 5 minuti </li></ul></li><li> Piastre con batteri uccisi UV possono essere conservati a testa in giù a 4 ° C per un massimo di 1 mese. </li></ol> Parte 2: Eseguire un uovo di tempo che per acquisire un età sincronizzato popolazione di animali In questa sezione abbiamo generare una popolazione di vermi con un comune tratteggiati data. Questo si ottiene agli adulti sessualmente attivi di deporre le uova su un piatto per un periodo definito di tempo e consentendo a tali uova di svilupparsi. <ol><li> (Opzionale) Trasferimento di circa 20 vermi adulti giovani per un piatto fresco senza FUDR NGM. Lasciare le piastre a 25 ° C al fine di consentire worm di propagarsi attraverso e mangiare tutto il cibo batterica. Dopo che il cibo dei batteri è stato consumato vermi appena schiusi entrerà nella fase di crescita arrestato dauer. Si inizierà a vedere la larva dauer circa entro una settimana, a seconda di quanti vermi si trasferisce alla piastra inizialmente. Dauer larva può essere utilizzato per i passi successivi per un massimo di 1 mese. </li><li> (Opzionale) Trasferimento 20-30 larva dauer ad una piastra NGM fresca senza FUDR. In presenza di larve cibo dauer diventeranno adulti sessualmente attivi entro 2 giorni e rimangono sessualmente attivi per alcuni giorni in seguito a 25 ° C. </li><li> Trasferimento 10-15 adulti sessualmente attivi ad una piastra NGM fresca senza FUDR. Questo piatto è denominato deposizione delle uova a tempo (TEL) piastra. </li><li> Lascia la piastra TEL a 25 ° C per 6 ore al fine di consentire tha tempo worm per deporre le uova. </li><li> Togliere i vermi adulti dal piatto TEL. Piastra può essere ispezionato visivamente per le uova prima di rimuovere gli adulti. Se il worm non hanno gettato un numero sufficiente di uova della piastra TEL può essere lasciato a 25 ° C fino a un totale di 24 ore prima di rimuovere i vermi adulti. </li><li> Luogo TEL piastra a 20 ° C fino a quando le uova si sono schiuse e il worm si sono sviluppati allo stadio larvale L4 (solitamente ciò richiede 2 giorni per tipo selvaggio C. elegans, ma può richiedere più tempo per i ceppi con fenotipo lento sviluppo). </li></ol> Parte 3: gli animali Punteggio per la durata della vita In questa sezione seguiamo l&#39;età sincronizzato popolazione di vermi da parte 2 fino alla morte. I worm sono mantenuti in Amp / FUDR piastre per prevenire la produzione di uova e di contaminazioni batteriche e sono considerati morti se non riescono a rispondere agli stimoli esterni. <ol><li> Trasferimento L4 larva a seminato Amp / FUDR piatti. Per ogni ceppo o condizione in fase di sperimentazione, è tipico di impostare 2-3 piatti con 25-30 vermi per piastra. Immagini della C. fasi della vita elegans sono forniti per riferimento (Figura 1) 1. </li><li> Luogo Amp / FUDR piastre a 20 ° C per 24 ore. </li><li> Dopo 24 ore, visivamente valutare i vermi, i media, e batteri. Trasferimento a fresco worm Amp / FUDR piastre se uno qualsiasi dei seguenti requisiti: <ul><li> I vermi hanno mangiato la maggior parte del cibo batterica. All&#39;inizio i vermi durata della vita esperimento dovrà essere trasferiti 1 a 2 volte la settimana per evitare che i batteri di essere impoverito. </li><li> Un numero significativo di larve sono presenti. Questo di solito è un&#39;indicazione che i vermi sono stati trasferiti al Amp / FUDR piatti come i giovani adulti, invece di L4s e sono stati in grado di porre alcune uova FUDR prima della entrata in vigore. Il FUDR generalmente impedisce alle larve di crescere in adulti a tariffa intera, ma a volte alcuni si svilupperà in adulti e si confondono con gli animali sperimentali. </li><li> Batteri vivi / crescita sono osservate. In generale, la combinazione di Amp e UV-uccisione farà in modo che i batteri non vivono contaminare l&#39;esperimento, ma di tanto in tanto si verifica. </li><li> Crescita fungina si osserva sui media. Se diagnosticato precocemente, la crescita fungina di solito può essere tagliato con l&#39;ausilio di un puntale o spatola. Una volta che cresce fino a essere più grande di pochi millimetri di diametro, di solito è più facile trasferire i vermi a un piatto nuovo. </li><li> Utilizzando la portata dissezione, determinare se ogni worm è vivo o morto. </li><li> Battere delicatamente la piastra. Il worm è vivo se si muove in risposta alle intercettazioni. </li><li> Se il worm non risponde al toccando l&#39;, zoom piastra sulla regione della testa. </li><li> Battere delicatamente la testa del verme con pick platino trasferimento. Punteggio ottenuto il worm come morto se non risponde muovendo la testa. </li><li> Vermi morti possono essere rimossi dalla piastra. </li></ul></li><li> Registrare la data e il numero di worm che sono vivi e morti. </li><li> Ritorna piastre a 20 ° C. </li><li> Ripetere i passi da 3.3 fino a 3,6 ogni 2 o 3 giorni fino a quando tutti i vermi sono morti. </li></ol> Parte 4: Risultati Rappresentante. I dati grezzi prodotti da un esperimento durata nematode la vita è un elenco di date con i numeri corrispondenti di vermi che sono vivi e morti per ogni ceppo testato. Il numero di worm che muoiono ogni giorno è tipicamente invertita per calcolare la percentuale di vermi vivi ogni giorno (Figura 2A), che è tracciata graficamente come una curva di sopravvivenza (Figura 2B, il giorno della deposizione delle uova tempo è considerato giorno 0 ). La durata per ogni singolo verme nello studio possono essere calcolati dal conteggio di vermi che muoiono ogni giorno e utilizzato per calcolare la durata della vita media e mediana per il confronto tra i ceppi. Il conteggio del numero di vermi vivi ogni giorno non viene utilizzato direttamente nell&#39;analisi durata della vita. Worms mantenuta su terreni solidi a volte &#39;fuga&#39;, o strisciare verso l&#39;alto la parete della piastra o giù di sotto della media. Il numero di vermi vivi ogni giorno può essere usato per determinare il numero di worm sono fuggiti in tutto il corso dell&#39;esperimento. Worm che fuggono sono tipicamente rimosso dall&#39;analisi. Come punto di riferimento, la durata della vita media tipica di N2, il C. elegans ceppo selvatico, mantenuti in UV-ucciso i batteri a 20 ° C è di circa 25 giorni, come misurato da un uovo. Parte 5: Soluzioni <table border="0"><tbody><tr><td colspan="2"> Nematode crescita Media (NGM), 100 ml: </td></tr><tr><td colspan="2"> Combina: </td></tr><tr><td> 0,3 g </td><td> NaCl </td></tr><tr><td> 0,25 g </td><td> Peptone </td></tr><tr><td> 2 g </td><td> Agar </td></tr><tr><td colspan="2"> Autoclave per 40 minuti e lasciare raffreddare a 55 ° C, quindi aggiungere: </td></tr><tr><td> 100 ul </td><td> 1 M MgSO 4 </td></tr><tr><td> 100 ul </td><td> 5 mg / ml colesterolo </td></tr><tr><td> 100 ul </td><td> 1 M CaCl2 </td></tr><tr><td> 1,625 ml </td><td> 1,5 M pH 6,0 Kpi </td></tr><tr><td colspan="2"> NGM liquido può essere utilizzato immediatamente a versare piatti o lascia solidificare e conservati a lungo termine a temperatura ambiente </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> Luria Broth (LB), 1L: </td></tr><tr><td> 10 g </td><td> BactoTryptone </td></tr><tr><td> 5 g </td><td> Estratto di Lievito </td></tr><tr><td> 10 g </td><td> NaCl </td></tr><tr><td> 10 mL </td><td> 1 M Tris pH 8.0 </td></tr><tr><td> 1 L </td><td> acqua deionizzata </td></tr><tr><td colspan="2"> Autoclave e conservare a temperatura ambiente. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1 M MgSO 4, 300 ml: </td></tr><tr><td> 73,95 g </td><td> MgSO 4 </td></tr><tr><td> 300 mL </td><td> acqua deionizzata </td></tr><tr><td colspan="2"> Autoclave e conservare a temperatura ambiente. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 5 mg / ml colesterolo, 200 ml: </td></tr><tr><td> 1 g </td><td> colesterolo </td></tr><tr><td> 200 mL </td><td> 100% di etanolo </td></tr><tr><td colspan="2"> Filtro sterilizzare e conservare a temperatura ambiente. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1 M CaCl 2, 500 ml: </td></tr><tr><td> 27,75 g </td><td> CaCl 2 </td></tr><tr><td> 500 ml </td><td> acqua deionizzata </td></tr><tr><td colspan="2"> Filtro sterilizzare e conservare a temperatura ambiente. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> KPI 1,5 M pH 6.0, 1L: </td></tr><tr><td colspan="2"> Combina: </td></tr><tr><td> 31,4 g </td><td> Kpi bibasico </td></tr><tr><td> 179,6 g </td><td> Kpi monobasico </td></tr><tr><td> 850 ml </td><td> acqua deionizzata </td></tr><tr><td colspan="2"> Calore durante la miscelazione per consentire KPI a dissolversi in soluzione. Regolare il pH a 6,0 con NaOH 10 N. </td></tr><tr><td colspan="2"> Aggiungere acqua deionizzata a 1 L. </td></tr><tr><td colspan="2"> Autoclave e conservare a temperatura ambiente. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1 M Tris, pH 8.0: </td></tr><tr><td> 60,57 g </td><td> Tris </td></tr><tr><td> 400 mL </td><td> acqua deionizzata </td></tr><tr><td colspan="2"> Regolare il pH a 8,0 con HCl. </td></tr><tr><td colspan="2"> Aggiungere acqua deionizzata a 500 ml. </td></tr><tr><td colspan="2"> Filtro sterilizzare e conservare a temperatura ambiente. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 150 mM Fluorodeoxyuridine (FUDR), 10 ml: </td></tr><tr><td> 0,3693 g </td><td> FUDR </td></tr><tr><td> 10 mL </td><td> sterile di acqua deionizzata </td></tr><tr><td colspan="2"> Conservare a -20 ° C. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 100 mg / ml Ampicillina (Amp), 10 ml: </td></tr><tr><td> 1 g </td><td> Ampicillina </td></tr><tr><td> 10 mL </td><td> sterile di acqua deionizzata </td></tr><tr><td colspan="2"> Conservare a -20 ° C. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 50 mg / ml Carbenicillin (CARB), 10 ml: </td></tr><tr><td> 500 mg </td><td> Carbenicillina </td></tr><tr><td> 10 mL </td><td> sterile di acqua deionizzata </td></tr><tr><td colspan="2"> Conservare a -20 ° C. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1 M Isopropyl β-d-Thiogalactopyranoside (IPTG), 10 ml: </td></tr><tr><td> 2,38 g </td><td> IPTG </td></tr><tr><td> 10 mL </td><td> sterile di acqua deionizzata </td></tr><tr><td colspan="2"> Conservare a -20 ° C. </td></tr></tbody></table><img src="/files/ftp_upload/1152/Figure%201.png" alt="figura 1" /> Immagini campo Figura 1. Brillante di C. fasi della vita elegans, tra cui uova, i quattro stadi larvali (L1 - L4) e adulti. Tutti i pannelli mostrano ermafroditi tranne in basso a destra, che mostra un maschio adulto (immagine da Wood (1988)). <img src="/files/ftp_upload/1152/Figure%202.png" alt="figura 2" /> I risultati rappresentativi Figura 2. Dal C. sperimentare la vita elegans durata confrontando ceppo selvatico N2 tipo di un ceppo che contiene una mutazione nel gene daf-2. (A) una tabella che mostra i dati raccolti, incluso il numero di giorni dal worm sono state le uova, il numero di vermi morti osservati ogni giorno, e la percentuale del campione originale rimanendo in vita ogni giorno (come calcolato dai conti ogni giorno di vermi morti) . (B) Sopravvivenza curve corrispondenti ai dati di durata della vita previsto (A). <imgsrc = &quot;/ files/ftp_upload/1152/Figure 3.jpg&quot; alt = &quot;Figura 3&quot; /&gt; Figura 3. Rappresentante di lipofuscina confronto tra adulti C. giovani e anziani elegans. Immagini in campo chiaro vengono visualizzati sul sinistro e canale di fluorescenza DAPI immagini sulla destra. I pannelli in alto mostrano un worm 4 giorni vecchi e pannelli inferiori mostrano un verme 11 giorni di età (come misurato da uova).

<ol>
	<li>Wood, W. B., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Nematode Caenorhabditis elegans. , 1-16 (1988).</li><li>Sutphin, G. L., Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+restriction+by+bacterial+deprivation+increases+life+span+in+wild-derived+nematodes.">Dietary restriction by bacterial deprivation increases life span in wild-derived nematodes.</a> Exp Gerontol. 43, 130-135 (2008).</li><li>Garigan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+tissue+aging+in+Caenorhabditis+elegans:+a+role+for+heat-shock+factor+and+bacterial+proliferation.">Genetic analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation.</a> Genetics. 161, 1101-1112 (2002).</li><li>Chen, D., Pan, K. Z., Palter, J. E., Kapahi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longevity+determined+by+developmental+arrest+genes+in+Caenorhabditis+elegans.">Longevity determined by developmental arrest genes in Caenorhabditis elegans.</a> Aging Cell. 6, 525-523 (2007).</li><li>Curran, S. P., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifespan+regulation+by+evolutionarily+conserved+genes+essential+for+viability.">Lifespan regulation by evolutionarily conserved genes essential for viability.</a> PLoS Genet. 3, e56-e56 (2007).</li><li>Dillin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rates+of+behavior+and+aging+specified+by+mitochondrial+function+during+development.">Rates of behavior and aging specified by mitochondrial function during development.</a> Science. 298, 2398-2401 (2002).</li><li>Hamilton, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+RNAi+screen+for+longevity+genes+in+C.+elegans.">A systematic RNAi screen for longevity genes in C. elegans.</a> Genes Dev. 19, 1544-1555 (2005).</li><li>Hansen, M., Hsu, A. L., Dillin, A., Kenyon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+genes+tied+to+endocrine,+metabolic,+and+dietary+regulation+of+lifespan+from+a+Caenorhabditis+elegans+genomic+RNAi+screen.">New genes tied to endocrine, metabolic, and dietary regulation of lifespan from a Caenorhabditis elegans genomic RNAi screen.</a> PLoS Genet. 1, 119-128 (2005).</li><li>Lee, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+RNAi+screen+identifies+a+critical+role+for+mitochondria+in+C.+elegans+longevity.">A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity.</a> Nat Genet. 33, 40-48 (2003).</li><li>Smith, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-+and+calorie-independent+life+span+extension+from+dietary+restriction+by+bacterial+deprivation+in+Caenorhabditis+elegans.">Age- and calorie-independent life span extension from dietary restriction by bacterial deprivation in Caenorhabditis elegans.</a> BMC Dev Biol. 8, 49-49 (2008).</li><li>Hodgkin, J., Horvitz, H. R., 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=Nondisjunction+Mutants+of+the+Nematode+CAENORHABDITIS+ELEGANS.">Nondisjunction Mutants of the Nematode CAENORHABDITIS ELEGANS.</a> Genetics. 91, 67-94 (1979).</li><li>Emmons, S. W., Sternberg, P. W., Riddle, D. L., Blumenthal, T., Meyer, B. J., Priess, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. ELEGANS II. , (1997).</li><li>Lipton, J., Kleemann, G., Ghosh, R., Lints, R., Emmons, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mate+searching+in+Caenorhabditis+elegans:+a+genetic+model+for+sex+drive+in+a+simple+invertebrate.">Mate searching in Caenorhabditis elegans: a genetic model for sex drive in a simple invertebrate.</a> J Neurosci. 24, 7427-7434 (2004).</li><li>Davis, B. O., Anderson, G. L., Dusenbery, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+luminescence+spectroscopy+of+fluorescence+changes+during+aging+in+Caenorhabditis+elegans.">Total luminescence spectroscopy of fluorescence changes during aging in Caenorhabditis elegans.</a> Biochemistry. 21, 4089-4095 (1982).</li><li>Klass, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+in+the+nematode+Caenorhabditis+elegans:+major+biological+and+environmental+factors+influencing+life+span.">Aging in the nematode Caenorhabditis elegans: major biological and environmental factors influencing life span.</a> Mech Ageing Dev. 6, 413-429 (1977).</li><li>Steinkraus, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+restriction+suppresses+proteotoxicity+and+enhances+longevity+by+an+hsf-1-dependent+mechanism+in+Caenorhabditis+elegans.">Dietary restriction suppresses proteotoxicity and enhances longevity by an hsf-1-dependent mechanism in Caenorhabditis elegans.</a> Aging Cell. 7, 394-404 (2008).</li></ol>

Il controllo genetico della longevità è stato studiato estensivamente in C. elegans, in gran parte dovuto alla facilità e rapidità con cui durata può essere determinata. Il protocollo descritto in questo articolo descrive un quadro di base per ottenere dati riproducibili durata della vita in C. elegans e può essere applicato anche alle specie relatednematode 2. Facendo alcune modifiche semplici queste procedure possono essere adattati per misurare la durata della vita sotto una variet?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Agar</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0145-17-0 (214530)</td>
<td>&nbsp;</td>
<tr>
<td>Ampicillin</td>
<td>Reagent</td>
<td>MidSci</td>
<td>0339</td>
<td>&nbsp;</td>
<tr>
<td>BactoTryptone</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0123-17-3 (211705)</td>
<td>&nbsp;</td>
<tr>
<td>BactoPeptone</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0118-17-0 (211677)</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>NC9699248 (1332-01)</td>
<td>&nbsp;</td>
<tr>
<td>Carbenicillin</td>
<td>Reagent</td>
<td>Gold BioTechnology (GBT)</td>
<td>C0109</td>
<td>&nbsp;</td>
<tr>
<td>Cholesterol</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>C75209</td>
<td>&nbsp;</td>
<tr>
<td>Fluorodeoxyuridine (FUDR)</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F0503</td>
<td>&nbsp;</td>
<tr>
<td>Isopropyl &#x03B2;-d-Thiogalactopyranoside (IPTG)</td>
<td>Reagent</td>
<td>Gold BioTechnology (GBT)</td>
<td>I2481C5</td>
<td>&nbsp;</td>
<tr>
<td>KPi Dibasic</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>5087862 (3252-01)</td>
<td>&nbsp;</td>
<tr>
<td>KPi Monobasic</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>5087861 (3246-01)</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>NC9561800 (2500-01)</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>S251</td>
<td>&nbsp;</td>
<tr>
<td>Tris</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0886-17-0 (288620)</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

measuring caenorhabditis elegans life span on solid media

L&#39;invecchiamento è un processo degenerativo caratterizzato da un progressivo deterioramento delle componenti cellulari e gli organelli con conseguente mortalità. Il nematode<em&gt; Caenorhabditis elegans</em&gt; È emerso come principale modello utilizzato per studiare la biologia dell&#39;invecchiamento. Perché praticamente ogni sottosistema biologica subisce declino funzionale con l&#39;aumentare dell&#39;età, la durata della vita è l&#39;obiettivo primario di interesse se si considera tasso totale di invecchiamento. Nei nematodi, la durata è tipicamente definito come il numero dei giorni di un animale rimane sensibile agli stimoli esterni. Nematodi possono essere propagati sia in mezzi liquidi o su un supporto solido in lastre, e sono state sviluppate tecniche per la misurazione della durata della vita in entrambe le condizioni. Qui vi presentiamo un protocollo generalizzato per la misurazione della durata di nematodi mantenuta su terreni solidi crescita nematodi e nutriti con una dieta a base di raggi UV-batteri uccisi. Queste procedure possono essere facilmente adattato alle durata del test vita sotto varie condizioni comuni, tra cui una dieta composta di batteri vivi, restrizione dietetica, e l&#39;interferenza dell&#39;RNA.

Watch this Scientific Journal Video about Measuring Caenorhabditis elegans Life Span on Solid Media at JoVE.com

Measuring Caenorhabditis elegans Life Span on Solid Media

In questo articolo vi presentiamo un protocollo generale per la misurazione della durata di nematodi mantenuto su un supporto solido con UV ucciso cibo batterica.

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 ...

measuring-caenorhabditis-elegans-life-span-on-solid-media

Research

JoVE Journal

Biology

Measuring Caenorhabditis elegans Life Span on Solid Media

29.7K Views.  University of Washington. In questo articolo vi presentiamo un protocollo generale per la misurazione della durata di nematodi mantenuto su un supporto solido con UV ucciso cibo batterica.

Video: Measuring Caenorhabditis elegans Life Span on Solid Media

Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and chronological life spans are two established paradigms used to study aging in yeast. Replicative aging is defined as the number of daughter cells a single yeast mother cell produces before senescence; chronological aging is defined by the length of time cells can survive in a non-dividing, quiescence-like state 2. We have developed a high-throughput method for quantitative measurement of chronological life span. This method involves aging the cells in a defined medium under agitation and at constant temperature. At each age-point, a sub-population of cells is removed from the aging culture and inoculated into rich growth medium. A high-resolution growth curve is then obtained for this sub-population of aged cells using a Bioscreen C MBR machine. An algorithm is then applied to determine the relative proportion of viable cells in each sub-population based on the growth kinetics at each age-point. This method requires substantially less time and resources compared to other chronological lifespan assays while maintaining reproducibility and precision. The high-throughput nature of this assay should allow for large-scale genetic and chemical screens to identify novel longevity modifiers for further testing in more complex organisms.

Chronological aging in yeast refers to the loss of cell viability associated with time in stationary phase. Here we describe a high-throughput method for quantitatively determining yeast chronological life span.

Quantificare cronologica Span lievito Life di conseguenza di cellule invecchiate

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and ...

Ricerca

Biologia

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

Durata pannello misura replicazione in lievito

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

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 ...

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 ...

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

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

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

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

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 ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Misura Stress ossidativo Resistenza<em&gt; Caenorhabditis elegans</em&gt; In 96 pozzetti micropiastre

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

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

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

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

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

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

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Imaging privo di etichette delle dinamiche di conservazione lipidica negli elegani di caenorhabditis utilizzando la microscopia a dispersione Raman stimolata

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

Automatizzare la quantificazione aggregata in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...