This research was supported by NIH Grant AG043080 and the National Natural Science Foundation of China (NSFC), No. 11434001. We thank Lucas Waldburger for proofreading the manuscript.

1. Silicon Wafer Mold Fabrication NOTA: La fotomaschera è stato progettato con il software AutoCAD e prodotto da una società commerciale. Questo disegno contiene tre strati di diversi modelli ( <a href="http://ecsource.jove.com/files/ftp_upload/55412/single_cell_trap.dwg" target="_blank">File supplementare 1</a> ). Le altezze di primo, secondo, e terzo strato sono circa 4 pm, 10 pm e 50 um, rispettivamente. Lo stampo wafer di silicio è stata creata dalla fotomaschera mediante litografia soft 9, 10. <ol><li> Cuocere un wafer di silicio a 200 ° C per 10 minuti per evaporare il vapore acqueo. cappotto centrifuga fotoresist negativo SU-8 sul wafer di silicio. NOTA: coat Spin SU-8 3005 a 5.000 giri al minuto per 30 s per generare il primo strato, SU-8 2010 a 3.000 rpm per 30 s per generare il secondo strato, e SU-8 3025 a 2.000 giri al minuto per 30 s per generare il terzo strato. </li><li> Soft-cuocere il wafer rivestito prima di tegli trasferimento modello. Allineare e esporre il wafer in modalità &quot;contatto diretto&quot; utilizzando un allineatore maschera. NOTA: Soft-cuocere il wafer per 2 min a 95 ° C per il primo strato, 3 min a 95 ° C per il secondo strato, e 15 min a 95 ° C per il terzo strato. Utilizzare la dose di esposizione a 100 mJ / cm 2 per il primo strato, 130 mJ / cm 2 per il secondo strato, e 200 mJ / cm 2 per il terzo strato. </li><li> Dopo l&#39;esposizione, post-cuocere il wafer e sviluppare utilizzando SU-8 sviluppatore. Essiccare il wafer utilizza una pistola N 2 e duro-cuocere il wafer a 200 ° C per 30 min. NOTA: Lo stampo wafer di silicio è fissata a 15 cm di diametro plastica Petri piatto con del nastro adesivo, con il lato rivolto verso l&#39;alto modello. Solitamente, abbiamo messo diverse strutture identiche di chip sullo stesso stampo che permette a più chip microfluidici per essere fabbricati contemporaneamente. Ogni stampo può essere riutilizzato molte volte per fabbricare chip microfluidici. </li></ol> 2. Microfluidicfabbricazione di chip <ol><li> Mettere una barca pesa pulita su una bilancia e tarare la bilancia. Versare 50 g di base di PDMS nella barca pesare. </li><li> Aggiungere 5 g di PDMS agente di indurimento alla barca pesare (w / w rapporto di 1:10 alla base PDMS). NOTA: Questo volume è basato su 15 cm con piastra Petri con lo stampo. Regolare la quantità di reagente se si utilizza una piastra di Petri di dimensioni diverse. </li><li> Mescolare la base PDMS e l&#39;agente di indurimento con una pipetta monouso. Iniziare dal bordo della barca di pesatura e si muovono lentamente verso l&#39;interno. Mescolare accuratamente per alcuni minuti fino formano piccole bolle nella miscela; accurata miscelazione è essenziale per PDMS polimerizzazione. </li><li> Versare il composto lentamente nella capsula di Petri; la miscela deve coprire completamente lo stampo wafer di silicio. </li><li> Porre la capsula di Petri in un vuoto per 10 minuti per rimuovere tutte le bolle d&#39;aria dalla miscela PDMS. Se le bolle rimangono sulla superficie della miscela, utilizzare una pipetta per far saltare fuori. </li><li> Incubare il siliciostampo wafer con PDMS in un forno a 75 ° C per circa 2 ore. </li><li> buccia delicatamente lo strato di PDMS polimerizzate fuori stampo wafer di silicio, evitando danni alla costruzione dello stampo wafer. In alternativa, tagliare il PDMS direttamente dallo stampo wafer di silicio con un margine minimo di 5 mm intorno al pattern usando una lama di rasoio industriale singolo bordo; buccia delicatamente lo strato PDMS dallo stampo wafer. </li><li> Posizionare il livello PDMS sul banco con il lato rivolto verso l&#39;alto modello. Asportare accuratamente il chip individuo con una lama di rasoio industriale singolo bordo, mantenendo un adeguato margine dal bordo di ogni singolo chip per evitare danni costruzione. </li><li> Con il lato rivolto verso l&#39;alto modello, utilizzare una penna punzone (ID 0,75 mm) per perforare verso il basso attraverso i cerchi ingresso e di uscita su ciascun lato dei canali. NOTA: Questi fori creano il percorso per il passaggio del fluido. Pertanto, è fondamentale passare attraverso i cerchi e punzone tutto il percorso attraverso lo strato PDMS. Assicurati cherimuovere le colonne PDMS dal foro. </li><li> Controllare ogni buco perforato inserendo di nuovo l&#39;ago penna pugno nel foro. Assicurarsi che l&#39;ago può uscire dall&#39;altra parte, che indica che non v&#39;è alcun blocco. Applicare del nastro alla superficie del modello, quindi rimuovere delicatamente il nastro per pulire le particelle di polvere. Ripetere questa operazione almeno tre volte. Lascia un pezzo pulito di nastro adesivo sui PDMS per mantenere la sterilizzazione. </li><li> Ripetere questa procedura sul lato opposto delle PDMS e lasciare l&#39;ultimo pezzo di nastro adesivo sulla pure. </li><li> Preparare un vetro di copertura 24 mm x 30 mm con uno spessore di 0.13-0.17 mm. Spruzzare etanolo al 70% sul vetro e secco con depolveratore per sterilizzare la superficie; Inoltre, il vetro può essere lavato con acqua ed essiccato in autoclave con depolveratore. </li><li> Trasferire il vetrino coprioggetto e PDMS ad una piastra di plastica. Rimuovere il nastro adesivo dal PDMS e posizionare il lato modello rivolto verso l&#39;alto. Evitare il contatto con la superficie del modello durante il trasferimento. </li><li&gt; Posizionare la piastra di plastica nella macchina plasma. Applicare un trattamento al plasma di ossigeno per il PDMS e il vetrino coprioggetto per rendere le superfici idrofile con i seguenti parametri di funzionamento: esposizione, 75 s; stabilizzazione gas, 20 cc / min; pressione, 200; e potenza, 100 W. </li><li> posizionare accuratamente il PDMS sul vetro di copertura, che collega entrambe le superfici idrofile (le superfici affrontate durante il trattamento al plasma). Assicurarsi che non ci siano bolle d&#39;aria tra il PDMS e il vetro di copertura. </li><li> Incubare il chip PDMS in un forno a 75 ° C per almeno 2 ore. NOTA: microfluidica carenti possono causare perdite di liquido sul microscopio durante l&#39;esperimento. Pertanto, è importante controllare il legame tra il PDMS e il vetro di copertura sollevando leggermente il PDMS dai bordi con pinzette. Delicatamente sollevando le PDMS non dovrebbero separarla dal vetro di copertura, che indica un legame di successo. </li></ol> 3. Preparazione per l&#39;esperimento <ol><li&gt; preparazione del tubo. <ol><li> Immergere i tubi di ingresso e uscita in soluzione di etanolo al 70% separatamente 1 giorno prima preparazione di chip PDMS. </li><li> Riempire una siringa da 5 ml con acqua in autoclave per lavare i tubi. Lavare ciascun tubo collegando il tubo sulla siringa e il lavaggio del tubo con acqua. </li><li> Ripetere la fase di lavaggio, almeno 3 volte per pulire residui etanolo. </li></ol></li><li> Esaminare i canali PDMS sotto un microscopio ottico con un obiettivo 10X per assicurarsi che la struttura è coerente e intatto. Stabilizzare il chip PDMS sulla piattaforma microscopio utilizzando nastro adesivo. NOTA: Fare chip multipli in una sola volta in fase 2 per garantire che non ci sono chip di backup, soprattutto se rendendo il dispositivo per la prima volta. </li><li> Riempire una siringa da 5 ml con acqua in autoclave. Fissare la siringa ad una pompa a siringa e inserire i tubi di ingresso e uscita corrispondenti. Impostare la velocità di lavaggio a 750 microlitri / h per risciacquare il chip per circa 10 min. Le bolle d&#39;aria sui canali ramodevono essere lavati via durante questo periodo; se vi sono bolle rimanenti, regolare manualmente la velocità per eliminare le bolle. </li><li> Lievito preparazione del campione. <ol><li> Trasferire 1 ml di campione preparato lievito (OD 600 0,6-0,8) in due provette da 1,5 ml microcentrifuga e centrifugare per 5 min a 3000 x g. </li><li> Rimuovere la maggioranza del surnatante da ogni provetta e combinare il resto per formare un campione da 0,5 mL. </li></ol></li><li> Sospendere la pompa a siringa e rimuovere i tubi di ingresso dal chip PDMS. </li><li> invertire manualmente la pompa a siringa per aspirare una piccola bolla d&#39;aria (1 a 2 cm di lunghezza) in ogni provetta di ingresso. Passare a succhiare nel campione di lievito; la bolla d&#39;aria mostrerà il confine campione e indicare la quantità di campione è stato elaborato. NOTA: Evitare di succhiare il campione nella siringa. </li><li> Inserire il tubo di ingresso posteriore nel chip PDMS e riavviare la pompa per caricare le cellule ad una velocità di 750 ml / h. </li><li> Lasciare la siringa pompa per 10 min lan ed esamina la progressione carico cella sotto il microscopio; un carico di successo sarà cellule intrappolare inferiore più del 60% di sospendere pilastro. </li><li> Passare alla soluzione di alimentazione e regolare la velocità a 400 microlitri / h per la coltura delle cellule. </li><li> posizioni di osservazione Select per il microscopio. NOTA: Per le misure di durata della vita, le immagini sono state scattate una volta ogni 15 minuti da un microscopio ottico con un obiettivo 40x. Per l&#39;analisi reporter fluorescente, immagini sono state prese una volta ogni 15 minuti con un microscopio a fluorescenza con un obiettivo olio 60X. </li></ol>

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	<li>Mortimer, R. K., Johnston, 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=Life+span+of+individual+yeast+cells.">Life span of individual yeast cells.</a> Nature. 183 (4677), 1751-1752 (1959).</li><li>Polymenis, M., Kennedy, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology:+High-tech+yeast+ageing.">Cell biology: High-tech yeast ageing.</a> Nature. 486 (7401), 37-38 (2012).</li><li>Xie, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+phenotyping+of+aging+in+single+yeast+cells+using+a+novel+microfluidic+device.">Molecular phenotyping of aging in single yeast cells using a novel microfluidic device.</a> Aging Cell. 11 (4), 599-606 (2012).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+analysis+of+yeast+replicative+aging+using+a+new+generation+of+microfluidic+device.">Single cell analysis of yeast replicative aging using a new generation of microfluidic device.</a> PLoS One. 7 (11), e48275 (2012).</li><li>Chen, K. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Xie, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+telomerase+inactivation+accelerates+aging+independently+of+telomere+length.">Early telomerase inactivation accelerates aging independently of telomere length.</a> Cell. 160 (5), 928-939 (2015).</li><li>Boy-Marcotte, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heat+shock+response+in+yeast:+differential+regulations+and+contributions+of+the+Msn2p/Msn4p+and+Hsf1p+regulons.">The heat shock response in yeast: differential regulations and contributions of the Msn2p/Msn4p and Hsf1p regulons.</a> Mol Microbiol. 33 (2), 274-283 (1999).</li><li>Yang, X., Lau, K. 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The authors declare that they have no competing financial interests.

Il dispositivo PDMS deve essere preparata. Altrimenti, le bolle d&#39;aria causate inserendo tubi nel dispositivo saranno difficili da rimuovere. Fase 3.4 è importante per migliorare l&#39;efficienza di carico delle cellule concentrando le cellule. Per aumentare la velocità dell&#39;esperimento, da 4 a 6 moduli sullo stesso chip PDMS collegati a pompe indipendentemente operativi sono tipicamente utilizzati per eseguire 4 a 6 diversi esperimenti (ceppi diversi o composizioni media) simultaneamente. <p class="jove_c...

Erba lievito è un potente organismo modello nella ricerca sull&#39;invecchiamento. Tuttavia, un test di durata della vita convenzionale nel lievito si basa su microdissezione, che non è solo lavoro intensivo, ma anche a basso throughput di 1, 2. Inoltre, l&#39;approccio tradizionale microdissezione non fornisce una vista dettagliata di varie caratteristiche cellulari e molecolari nelle cellule madri singole che invecchiano. Lo sviluppo di dispositivi microfluidici ha permesso una procedura automatizzata per misurare durata lievito nonché a seguire marcatori molecolari e diversi fenotipi cellulari per tutta la durata delle cellule madri 3, 4, 5, 6, 7, 8. Dopo che le cellule di lievito vengono caricati in un dispositivo microfluidico, possono essere monitorati sotto un microscopio utilizzando temporali giri automaticie di imaging. Con l&#39;aiuto di strumenti di elaborazione di imaging, diversi fenotipi cellulari e molecolari possono essere estratti 3, 6, 8, compresa durata, dimensioni, reporter fluorescente, morfologia cellulare, le dinamiche del ciclo cellulare, ecc, molti dei quali sono difficili o impossibili da ottenere utilizzando il metodo microdissezione tradizionale. Dispositivi microfluidici hanno assunto un rilievo nella ricerca affinamento sui lieviti in quanto il loro sviluppo di successo di qualche anno fa 3, 4, 6, 7. Diversi gruppi hanno pubblicato successivamente sulle variazioni dei disegni precedenti 5, e molti laboratori di lievito hanno impiegato dispositivi microfluidici per il loro studio. In una cultura di cellule in fase di crescita esponenziale, il numero di cellule madri di età compresa tra che sono disponibili per l&#39;osservazione è miniscule. Pertanto, il principio di progettazione generale del dispositivo microfluidica per le misure di durata della vita è quello di mantenere le cellule madre e per rimuovere le cellule figlie. Uno di questi disegni si avvale del fatto che il lievito subisce divisione cellulare asimmetrica. Le strutture a cellule madri trappola dispositivo volontà grandi e permettono cellule figlie piccole per essere lavato via. Il chip microfluidico descritto in questo articolo utilizza un tampone morbido polidimetilsilossano (PDMS) (colonne pensili verticali) per intrappolare cellule madri (Figura 1). Dispositivi di tipo analogo sono stati riportati precedentemente 3, 4, 6, 7. Questo protocollo utilizza una procedura semplice da fabbricare dispositivi microfluidici e un metodo cella di carico semplice che è ottimizzato per gli esperimenti time-lapse imaging. Uno dei parametri chiave del dispositivo microfluidico è la larghezza delle pastiglie PDMS utilizzati per cellule madri trappola. Il nostro device usa pastiglie più ampi che possono tenere più cellule madri sotto ogni pad, tra cui una frazione significativa di cellule madri fresche che possono essere monitorati per tutta la loro durata di vita. Oltre alle misurazioni durata della vita, questo protocollo è utile per esperimenti di imaging singola cellula time lapse quando le cellule devono essere monitorati per molte generazioni o quando un&#39;osservazione tutta la durata della vita è necessaria.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3&#39;&#39; &#60;111&#62; silicon wafer</td>
			<td>Addison Engineering</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2000 and 3000 Series</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYLGARD&#174; 184 SILICONE ELASTOMER KIT</td>
			<td>ellsworth</td>
			<td>2065622</td>
			<td>Include Sylgard&#174; silicone elastomer base and curing agent</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>VWR</td>
			<td>391-1502</td>
			<td></td>
		</tr>
		<tr>
			<td>Harris Uni-core&#8482; punch(I.D. 0.75 mm)</td>
			<td>Sigma-Aldrich</td>
			<td>29002513</td>
			<td></td>
		</tr>
		<tr>
			<td>24 mm x 40 mm SLIP-RITE&#174; cover glass</td>
			<td>Thermo Fisher Scientific</td>
			<td>102440</td>
			<td></td>
		</tr>
		<tr>
			<td>3M&#160; Scotch Tape</td>
			<td>ULINE</td>
			<td>S-10223</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR&#174; Razor Blades</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td></td>
		</tr>
		<tr>
			<td>PURE ETHANOL, KOPTEC</td>
			<td>VWR</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>WHOOSH-DUSTER&#8482;</td>
			<td>VWR</td>
			<td>16650-027</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL BD Syringe (Luer-Lock&#8482;Tip)</td>
			<td>Becton, Dickinson and Company.</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE Standard Wall Tubing (100ft, AWG Size:22, Nominal ID: 0.028)</td>
			<td>COMPONENT SUPPLY COMPANY</td>
			<td>SWTT-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle Assortment</td>
			<td>COMPONENT SUPPLY COMPANY</td>
			<td>NEKIT-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>HACH</td>
			<td>2238300</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Oven</td>
			<td>FISHER SCIENTIFIC</td>
			<td>13246516GAQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon TE2000 microscope with 40x and 60x objective</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axio Observer Z1 with 40x and 60x objective</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>Longerpump</td>
			<td>TS-1B</td>
			<td></td>
		</tr>
	</tbody>
</table>

using microfluidic devices to measure lifespan and cellular phenotypes in single budding yeast cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

Dopo gli esperimenti, le durate di vita di cellule e molti fenotipi cellulari e molecolari possono essere estratti dalle immagini time-lapse registrate. Poiché ci sono un certo numero di diverse caratteristiche che possono essere estratti da ciascuna cella, il primo passo dell&#39;analisi è di annotare le cellule ed eventi, tra le posizioni ei confini delle celle e la tempistica dei vari eventi che vengono monitorati, quali come gli eventi in erba. Queste annotazioni che renderà più ...

Watch this Scientific Journal Video about Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells at JoVE.com

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Questo articolo presenta un protocollo ottimizzato per la produzione di chip microfluidici e la configurazione degli esperimenti microfluidici per misurare la durata di vita e fenotipi cellulari delle singole cellule di lievito.

Uso di dispositivi microfluidici misurare la durata della vita e cellulare fenotipi in singole cellule di lievito in erba

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

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Biology

7.6K Views.  Peking University.  Questo articolo presenta un protocollo ottimizzato per la produzione di chip microfluidici e la configurazione degli esperimenti microfluidici per misurare la durata di vita e fenotipi cellulari delle singole cellule di lievito. 

Video: Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

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 ...

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Biologia

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi di screening per identificare fenotipi postembrionale in C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Misura della durata della vita in<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

L&#39;acquisizione di fluorescenza filmati time-lapse di lievito in erba e Analisi Dinamica singola cella utilizzando innesti

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

La quantificazione temporale di espressione MAPK indotta in cellule di lievito singole

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

Su larga scala Knockdown Gene in<em&gt; C. elegans</em&gt; Utilizzo di librerie di alimentazione dsRNA per generare fenotipi Robusti Perdita di funzione

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Germogliamento lievito proteine ​​estrazione e purificazione per lo Studio della funzione, interazioni e modificazioni post-traduzionali

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

Le domande di pHluorin per quantitativa, Cinetica e high-throughput analisi di endocitosi in germogliamento lievito

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

Analisi spazio-temporale di Cytokinetic Eventi in lievito di fissione

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Ad alta risoluzione Imaging e analisi dei singoli microtubuli astrali Dynamics in germogliamento lievito

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...