Name: measuring oxidative stress resistance of caenorhabditis elegans in 96-well microtiter plates
Uploaded: 2015-05-09T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates at JoVE.com

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><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 60 x15 mm</td><td>Fisher</td><td>FB0875713A</td><td></td></tr><tr><td>Pipet 10 ml</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>Discovery v8 stereo zeiss microscope</td><td></td><td></td><td></td></tr><tr><td>96 well clear microtiter plate</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;flcn-1&lt;/em&gt; RNAi source</td><td>Ahringer Library</td><td></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

JoVE Journal

Biology

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

15.4K 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

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Un metodo semplice per High Throughput Screening chimico in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Ricerca

Biochimica

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

Piastra-base di coltivazione su larga scala di Caenorhabditis elegans: preparazione del campione per la studio di alterazioni metaboliche nel diabete

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

Rilevamento di metodi a basso costo per misurare la durata della vita e la durata della salute in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

Genetica

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.

Studiando lo Stress ossidativo causato dagli streptococchi di gruppo Mitis in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

Biologia dello sviluppo

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 ...

Biologia

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 ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

Misurazioni delle risposte allo stress fisiologico in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

Quantificazione del declino proteostatico tessuto-specifico nella Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on host physiology. It also fluoresces in blue when dying, providing a convenient means of pinpointing death. This property has been exploited to develop high-throughput label-free C. elegans survival assays (LFASS). These involve time-lapse fluorescence recording of worm populations set in multiwell plates, from which population median time of death can be derived. The present study adopts the LFASS approach to screen multiple microbial isolates at once for the effects on C. elegans susceptibility to severe heat and oxidative stresses. Such microbial screening pipeline, which can notably be used to prescreen probiotics, using severe stress resistance as a proxy for host health is reported here. The protocol describes how to grow both C. elegans gut microbiota isolate collections and synchronous worm populations in multiwell arrays before combining them for the assays. The example provided covers the testing of 47 bacterial isolates and one control strain on two worm strains, in two stress assays in parallel. However, the approach pipeline is readily scalable and applicable to the screening of many other modalities. Thus, it provides a versatile setup to rapidly survey a multiparametric landscape of biological and biochemical conditions that impact C. elegans health.

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

Gut microbes may positively or negatively impact the health of their host via specific or conserved mechanisms. Caenorhabditis elegans is a convenient platform to screen for such microbes. The present protocol describes high-throughput screening of 48 bacterial isolates for impact on nematode stress resistance, used as a proxy for worm health.

Screening ad alta produttività di isolati microbici con impatto sulla salute di Caenorhabditis elegans

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on ...

C. elegans Survival Assay: A Short-Term Toxicity Test

Source: Possik, E. and Pause, A. <a href="https://www.jove.com/video/2496/measuring-caenorhabditis-elegans-life-span-96-well-microtiter">Measuring Oxidative Stress Resistance of&#160;Caenorhabditis elegans&#160;in 96-well Microtiter Plates.</a>&#160;J. Vis. Exp. (2015).
This video introduces a short-term survival assay that tests a treatment for acute toxicity or tests a genetic strain for resistance to a toxic condition.&#160; The example protocol shows an example survival assay in which worms are tested for their ability to&#160; resist oxidative stress induced by paraquat (PQ).

C. elegans Test di sopravvivenza: un test di tossicità a breve termine

Source: Possik, E. and Pause, A. Measuring Oxidative Stress Resistance of&#160;Caenorhabditis elegans&#160;in 96-well Microtiter Plates.&#160;J. Vis. Exp. ...

Misura Stress ossidativo Resistenza

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