Riconosciamo il Centro Caenorhabditis Genetica per C. ceppi elegans. Supporto Il finanziamento è stato fornito dal Terry Fox Research Institute. Riconosciamo inoltre sostegno concesso al Parlamento europeo dal Rolande e Marcel Gosselin Graduate Studentship e la borsa di formazione CIHR / FRSQ in FRN53888 ricerca sul cancro del Programma di Formazione Cancer Research McGill integrato.

1. Preparazione dei reagenti <ol><li> Preparazione dei supporti per C. crescita elegans (in questo caso, gli animali wild-type e animali flcn-1 (ok975) mutante). <ol><li> Preparare Modified miscela secca di Youngren Solo Bacto-peptone (MYOB) contenente 5,5 g di Tris HCl, 2,4 g di Tris base, 31 g di Bactopetone, 20 g di NaCl e 0,08 g di colesterolo. Mescolare bene con agitazione. NOTA: Questo mix è sufficiente per preparare 10 litri di MYOB media. NOTA: mezzo di crescita (NGM) piastre normali potrebbero essere utilizzati al posto delle lastre MYOB 27. Tuttavia, lo stesso tipo di piastre deve essere utilizzato per una valida comparazione dei ceppi e tra ripetizioni indipendenti. In questo protocollo, abbiamo usato solo piatti MYOB. </li><li> Preparare 1 L di MYOB medio aggiungendo 6 g di MYOB mix secco, 17 g di agar e portare a 1 L con H 2 O e autoclave per 45 minuti a 122 ° C. Versare in piastre e lasciare asciugare a temperatura ambiente per 2 giorni. </li><li> Usando una tecnica sterile, inoculare 100 ml di culture di terreno LB brodo con E. batteri coli OP50 e crescono O / N a 37 ° C. </li><li> Piastre Seed MYOB con E. batteri coli OP50. Permettono di crescere a temperatura ambiente per 48 ore. </li></ol></li><li> Preparazione per la down-regulation gene utilizzando RNAi <ol><li> Preparare MYOB piastre RNAi-alimentazione. Procedere come nei passaggi 1.1.1 e 1.1.2. Dopo la sterilizzazione in autoclave, lasciare il mezzo di raffreddare fino a circa 55 ° C e aggiungere 1 ml di 1 M isopropilico β-D-1-thiogalactopyranoside (IPTG) e 1 ml di ampicillina 100 mm. </li><li> Streak E. coli HT115 batteri trasformati con un plasmide che esprime sia EV controllo o flcn-1 RNAi specifico ampicillina (50 ug / ml) / tetraciclina (15 ug / ml) piastre di agar. Grow cultura O / N a 37 ° C. </li><li> Scegli colonie e inoculare E. coli HT115 batteri trasformati con un plasmide che esprime o EV controllo o flcn-1 specifica RNAi e crescere la cultura in LB / ampicillina (50 mg / ml) di media e O / N a 37 &amp; #176; C. </li><li> Piatti di semi con E. coli HT115 EV batteri o HT115 flcn-1 batteri RNAi. NOTA: Mantenere le piastre 48 ore a temperatura ambiente prima dell&#39;uso. Se RNAi atterramento da alimentazione è efficace, utilizzare altri metodi di consegna del ds-RNA 28. </li></ol></li><li> Indurre stress ossidativo con paraquat (PQ): <ol><li> Preparare tampone M9 mescolando 3 g di KH 2 PO 4, 6 g di Na 2 HPO 4, 5 g di NaCl e 0,25 g di MgSO4 · 7 H 2 O. Portare a 1 litro con acqua distillata. Soluzione Autoclave per 45 minuti a 122 ° C e conservare le bottiglie a temperatura ambiente. </li><li> Il giorno del test, preparare la soluzione M9 / PQ contenenti. Per questo protocollo, utilizzare PQ 100 mm (0,1028 g di PQ sciolti in 4 ml di tampone M9 riempie un intero 96 pozzetti con 40 ml di M9 / PQ per pozzetto). ATTENZIONE: PQ è un composto pericoloso e altamente tossico. Maneggiare secondo le linee guida appropriate. NOTA: La prima volta che l&#39;esecuzione di un nuovo streno o una nuova condizione, si raccomanda di saggiare la sopravvivenza in concentrazioni crescenti di PQ per determinare la migliore concentrazione da utilizzare per uccidere gli animali entro 7-10 ore. </li></ol></li></ol> 2. Preparazione di Age-sincronizzato L4 Popolazione <ol><li> Trasferimento 20 ermafroditi adulte gravide ad una piastra MYOB fresco seminato con E. batteri coli OP50. Preparare più matrici in base al numero di vermi necessari durante il dosaggio. </li><li> Consentire loro di deporre le uova per 6 ore e quindi utilizzando un verme filo di platino selezionamento rimuovere le madri. Controllare le piastre al microscopio per valutare il numero di uova deposte. </li><li> Lasciare le uova per schiudersi e crescere a 20 ° C per 48 ore. A questo punto, gli animali sono in un L4 / giovane tardivamente adulti. Scegli stessi animali palco e trasferire ai pozzetti-soluzione contenente PQ. NOTA: Dal momento che 48 ore di incubazione si applica solo ai mutanti che crescono in modo simile a wild-type animali, è importante monitorare sviluppotasso di mutanti recentemente testati per garantire la conformità al tasso di sviluppo wild-type, in caso contrario, devono essere utilizzati altre linee temporali. </li><li> In alternativa, utilizzare tecniche di sincronizzazione. Genera popolazione sincronizzato di L1 vermi larve con una soluzione di ipoclorito (25 ml di acqua, 125 ml di 5,25% di ipoclorito di sodio, 50 ml di 4 M NaOH; avvolgere bottiglia con un foglio di alluminio per isolare dalla luce). Tuttavia, per questa analisi, non è consigliabile, in quanto intenso candeggio potrebbe influenzare il comportamento degli animali durante il dosaggio. ATTENZIONE: Maneggiare soluzione di ipoclorito secondo le linee guida. NOTA: quando si utilizza RNAi per regolare lungo il gene di interesse, sincronizzare gli ermafroditi come descritto nel paragrafo 2, con l&#39;eccezione del trasferimento delle madri alle piastre RNAi seminate con specifici batteri RNAi. Quando il colpo con l&#39;RNAi è debole, si raccomanda di alimentazione per più generazioni. </li></ol> 3. Esecuzione del Stress Ossidativo Resistenza Assay &lt;/ P&gt; <ol><li> Dispensare 40 ml di soluzione di 100 PQ mm (preparato fresco) per ogni pozzetto di una piastra da 96 pozzetti. Utilizzare almeno 12 pozzetti come repliche di ogni condizione (trattamento, mutante, etc.). Utilizzando un verme scelta filo di platino, trasferire 5-8 animali L4 larve di ogni bene che indica l&#39;ora di inizio e l&#39;ora di fine. </li><li> Quando si avvia un&#39;altra condizione, nota l&#39;ora di inizio e l&#39;ora di fine. Posizionare la piastra a 20 ° C nel tempo di incubazione se trasferire i vermi e segnando animali morti un&#39;ora dopo dal momento di inizio. </li><li> Utilizzando il microscopio da dissezione, contare la sopravvivenza ogni ora. Agitare delicatamente la piastra prima di iniziare a contare. Indicare i vermi che non si muovono, anche dopo avvistare una luce ad alta intensità, come animali morti. Indicare anche il numero di animali vivi. NOTA: Guardando forma verme, le code e il movimento della testa ad alto ingrandimento, contribuire a determinare vermi morti dal vivo. </li><li> Ripetere questa operazione fino a quando la maggior parte ogni ora vermi sono morti. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 4. Determinazione della sopravvivenza Percentuale in Every Time Point <ol><li> Calcolare il numero totale di animali per condizione sommando il numero totale di animali trasferiti ad ogni pozzetto. Ignorare gli animali che sono danneggiati o uccisi al momento del trasferimento. </li><li> Per ogni punto di tempo, calcolare il numero totale di animali morti per condizioni (somma di animali morti nei 12 pozzetti). Per ogni punto di tempo, di calcolare la percentuale di morte (numero di animali morti diviso per il numero totale degli animali e moltiplicato per 100) e la percentuale di sopravvivenza (100 meno cento morte). </li><li> Ripetere questo test almeno 3 volte indipendenti. </li></ol>

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The authors declare that they have no competing financial interests.

C. elegans è un interessante organismo modello per studiare la resistenza allo stress ossidativo in vivo geneticamente poiché può essere facilmente colto, e rapidamente conduce a un gran numero di prole geneticamente identici. Diversi metodi per misurare la resistenza allo stress ossidativo sono stati precedentemente descritti e sono basati sulla integrazione di piastre di coltura con le varie fonti di ROS quali PQ, rotenone, H 2 O 2, e juglone 25,26,29-32. Qui si de...

A reference was corrected in the publication of <a href="http://www.jove.com/video/52746">Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates</a>. There was an error with reference 34 in the Citations section. It has been updated from:
Allen, E., Walters, I.B., Hanahan, D. Brivanib a dual FGF/VEGF inhibitor,is active both first and second line againstmouse pancreatic neuroendocrine tumors developing adaptive/evasive resistance toVEGF inhibition. Clin Cancer Res. 17, (16) 5299-310 (2011).
to:
Allen, A. T., Maher, K. N., Wani, K. A., Betts, K. E., &#38; Chase, D. L. Coexpressed D1- and D2-like dopamine receptors antagonistically modulate acetylcholine release in Caenorhabditis elegans. Genetics. 188, 579-590 (2011).

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

Negli eucarioti, fosforilazione ossidativa che si svolgono nella catena di trasporto degli elettroni dei mitocondri è il principale motore della produzione di energia sotto forma di ATP. Le specie reattive dell&#39;ossigeno (ROS) sono un sottoprodotto naturale di questo processo. Nonostante il loro importante ruolo di molecole di segnalazione, eccessivo ROS può causare danni al DNA, proteine ​​carbonilazione, e l&#39;ossidazione dei lipidi. Uno squilibrio tra la produzione di ROS e la disintossicazione è causa di stress ossidativo, che porta alla perdita di energia, danno cellulare, e fa scattare la morte delle cellule 1,2. Lo stress ossidativo contribuisce all&#39;invecchiamento e allo sviluppo di molte malattie mortali tra cui il cancro, il diabete, malattie cardiovascolari e neurodegenerative 3-9. Le cellule si sono evolute strategie di difesa enzimatici e non enzimatici per mantenere livelli adeguati di ROS e di proteggere i loro elettori contro il danno ossidativo 1,2. Superossido dismutasi (SOD) Gli enzimi agiscono in primo luogo a convert superossido da H 2 O 2, che viene poi convertito in acqua catalasi o enzimi perossidasi. Strategie non enzimatici di difesa sono per lo più le molecole che reagiscono più velocemente con ROS rispetto alle macromolecole cellulari, proteggendo componenti cellulari essenziali. Nonostante il ruolo protettivo dei ROS enzimi disintossicanti, alcune molecole ROS sfuggire i meccanismi di difesa antiossidanti e portano al danno ossidativo. Detection, la riparazione e la degradazione dei componenti cellulari danneggiate sono strategie essenziali della difesa durante lo stress ossidativo 1,2. Segnalazione meccanismi coinvolti nella resistenza allo stress e lo stress ossidativo in particolare sono molto evolutivamente conservati 10,11. A differenza delle cellule esperimenti di coltura in cui le condizioni organismal sono solo parzialmente riprodotti, lo studio dello stress ossidativo in organismi modello 12,13 ha un grande significato. C. elegans è un nematode vita libera che può essere facilmente ed economicamente culturato su un prato batterica sui terreni di coltura. E &#39;di piccole dimensioni (circa 1 mm di lunghezza) e normalmente cresce come un ermafrodita sé concimazione, che facilita le manipolazioni genetiche. Ha un rapido ciclo di vita e una capacità riproduttiva alto, producendo circa 300 figli per ogni generazione, che lo rende uno strumento potente per eseguire grandi schermi genetiche 14. Le C. elegans genoma è completamente sequenziato e il 40-50% dei geni sono previsto per essere omologhi di geni associati alla malattia umana 15-18. L&#39;atterramento di geni di interesse utilizzando RNAi è rapido e facile in C. elegans. Gene giù regolamento potrebbe essere realizzato nutrire gli animali E. coli, batteri che ospitano un plasmide che esprime l&#39;RNA a doppio filamento che gli obiettivi del mRNA di interessi 19. Pertanto, la determinazione della funzione del gene utilizzando su larga scala schermi RNAi ha un grande impatto sulla comprensione delle malattie umane tra cui il cancro 20,21. Studi di oresistenza allo stress xidative in C. elegans hanno portato all&#39;identificazione di meccanismi conservati di resistenza allo stress ossidativo 13,22. Alcune vie individuate sono percorsi comuni che modulano la longevità e resistenza ad altre sollecitazioni così come ipossia, calore, e lo stress osmotico. Questi percorsi comprendono la segnalazione dell&#39;insulina, segnalazione TOR, e autofagia. Altre vie principali riguardano la disintossicazione del ROS come gli enzimi superossido dismutasi e degli enzimi catalasi, o riparare i danni come shock termico e chaperone proteine ​​11,13,22. Questo protocollo descrive come determinare la resistenza allo stress ossidativo di C. elegans a liquido. Abbiamo usato flcn-1 (ok975) e wild-type animali per dimostrare il protocollo dal momento che abbiamo già mostrato una maggiore resistenza allo stress ossidativo in caso di perdita di flcn-1 (ok975) in C. elegans 23. Abbiamo anche dimostrato che questo aumento della resistenza dipendesu AMPK e autofagia, un asse di segnalazione che migliora bioenergetica cellulare e favorisce la resistenza allo stress 23. PQ è un fattore di stress ossidativo che interferisce con la catena di trasporto degli elettroni per produrre specie di ossigeno reattive 24. Lo stesso dosaggio potrebbe essere adattato e altre fonti di ROS o composti che generano ROS potrebbe essere utilizzato come H 2 O 2 e rotenone. Saggi simili sono stati sviluppati su piastre in cui sono utilizzati basse concentrazioni di PQ 25,26. Il vantaggio di questo test è che è molto veloce, ed i risultati potrebbero essere ottenuti in un giorno. Inoltre, il volume totale di liquido utilizzato per eseguire il test di resistenza allo stress ossidativo in piastre da 96 pozzetti è bassa rispetto al volume usato per preparare PQ contenenti piastre. Pertanto, la quantità di PQ usata è nel dosaggio liquido è basso, il che rende il test poco costoso e limita la produzione di rifiuti tossici. Tuttavia, le limitazioni di questo test rispetto ai saggi piastra comprendono lack di cibo nel dosaggio liquido e la minore concentrazione di ossigeno nel liquido rispetto all&#39;aria. Questi sono fattori importanti che in alcuni casi, potrebbe influenzare i risultati. Pertanto, confermando riproducibilità utilizzando altri metodi di resistenza stress ossidativo è raccomandato per appoggiare risultati ottenuti in questa analisi.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Agar bacteriological grade</td>
			<td>Multicell</td>
			<td>800-010-LG</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriological peptone</td>
			<td>Oxoid</td>
			<td>LP0037</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride biotechnology grade</td>
			<td>Bioshop</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma</td>
			<td>C8503-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure tris hydrochloride</td>
			<td>Invitrogen</td>
			<td>15506-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris aminomethane</td>
			<td>Bio Basic Canada Inc</td>
			<td>77-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-202185A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Bioshop</td>
			<td>69-52-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Bio Basic Inc.</td>
			<td>8013-01-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl viologen dichloride hydrate</td>
			<td>Aldrich chemistry</td>
			<td>856177-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 60x15mm</td>
			<td>Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet 10ml</td>
			<td>Fisher</td>
			<td>1367520</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>G-Biosciences</td>
			<td>RC-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma</td>
			<td>M-5921</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Bioshop</td>
			<td>7558-79-4</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Discovery v8 stereo zeiss microscope</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 well clear microtiter plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>flcn-1 RNAi source</td>
			<td colspan="2">Ahringer Library</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring oxidative stress resistance of caenorhabditis elegans in 96-well microtiter plates

Lo stress ossidativo, che è il risultato di uno squilibrio tra la produzione e la detossificazione di specie reattive dell&#39;ossigeno, è un fattore importante di patologie umane croniche, tra cui malattie cardiovascolari e neurodegenerative, diabete, invecchiamento e cancro. Pertanto, è importante studiare stress ossidativo non solo in sistemi cellulari ma anche utilizzando interi organismi. C. elegans è un interessante organismo modello per studiare la genetica di ossidative vie di trasduzione del segnale di stress, che sono altamente evolutivamente conservate. Qui, mettiamo a disposizione un protocollo per misurare la resistenza allo stress ossidativo in C. elegans a liquido. Brevemente, ROS inducono reattivi come paraquat (PQ) e H 2 O 2 vengono sciolti in tampone M9, e soluzioni sono aliquotati nei pozzetti di una piastra microtiter a 96 pozzetti. L4 sincronizzato / ragazzo C. animali elegans sono trasferiti nei pozzetti (5-8 animali / e) e la sopravvivenza è misurataogni ora fino maggior parte dei worm sono morti. Quando si esegue un test di resistenza allo stress ossidativo con una bassa concentrazione di fattori di stress in lastre, l&#39;invecchiamento potrebbe influenzare il comportamento degli animali su stress ossidativo, che potrebbero portare ad una interpretazione errata dei dati. Tuttavia, nel saggio qui descritto, questo problema è improbabile che si verifichi in quanto vengono utilizzati solo L4 / giovani adulti. Inoltre, questo protocollo è poco costoso e risultati sono ottenuti in un giorno, che rende questa tecnica attraente per schermi genetici. Nel complesso, questo aiuterà a capire ossidativi vie di segnale di stress di trasduzione, che si potrebbe tradurre in una migliore caratterizzazione delle ossidativo malattie umane allo stress associato.

Confrontando wild-type C. elegans per flcn-1 (ok975) animali mutanti Qui abbiamo usato PQ 100 mM per determinare la resistenza di tipo selvaggio C. elegans animali rispetto a flcn-1 (ok975), che ha dimostrato di resistere allo stress ossidativo, il calore, e anossia 23. Dopo 4 ore di trattamento, il 48,3% di wild-type sopravvissuto rispetto al 77,8% di sopravvivenza in flcn-1 (ok975) animali. Come previsto, flcn-1 (ok975) anima...

Watch this Scientific Journal Video about Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates at JoVE.com

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 ...

measuring-oxidative-stress-resistance-caenorhabditis-elegans-96-well

Research

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Biology

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

15.3K Views.  McGill University. 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. 

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

Measuring Caenorhabditis elegans Life Span on Solid Media

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

Measuring Caenorhabditis elegans Life Span on Solid Media

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

Misura Caenorhabditis elegans Life Span su terreni solidi

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

Ricerca

Biologia

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

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

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

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

Isolamento e In vitro Attivazione di Caenorhabditis elegans Sperm

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

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 ...

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 ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

Ice-Cap: Un metodo per la Crescita Arabidopsis E piante di pomodoro in piastre con 96 pozzetti di High-Throughput Genotipizzazione

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

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, ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

Analisi dello Stress Ossidativo in embrioni di zebrafish

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

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

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

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 ...