La ricerca è stata finanziata dal NIH 1R15GM094770.

1. Colonia di isolamento e di fissaggio <ol><li> Incubare 300 colonie su agar per il tempo indicato (una colonia isolata dovrebbe essere 1-2 mm di diametro). </li><li> Rimuovere colonia (faccia in su) e medie di fondo con una spatola stretta. </li><li> Mettere alcune gocce del 2% agar 42 ° C su un vetrino da microscopio utilizzando 1 ml Pipetman punta e subito colonia posto sul volto di agar prima si solidifica. </li><li> Mettere alcune gocce del 2% agar 42 ° C in colonia e lasciare solidificare. </li><li> Indossare guanti per tutte le fasi per il resto di questo protocollo </li><li> Blocco assetto con lama di rasoio e metterli in un mL 3,5 borosilicato con tappo a vite fiala contenente 2% paraformaldeide / fissativo glutaraldeide 2% per 7 giorni a 4 ° C. </li></ol> 2. Lavaggi e trattamenti Osmio <ol><li> Dopo circa 1 settimana, lavare blocchi di agar su ghiaccio sotto cappa incubando per 15 minuti due volte con abbastanza cacodilato di sodio 0,15 m (pH 7,2) per coprire completamente la colonia (circa 1,5 ml), poi altre due volte per 5 minuti con lo stesso volume di OS 1X tampone (100 mM KH 2 PO 4 10 mM MgCl 2, pH 6,0). </li><li> Aggiungere lo stesso volume di 1% OsO 4 [2 ml OsO 4 (2%) + 2 ml 2x OS Buffer] per fiale (sul ghiaccio) per coprire i blocchi agar sotto cappa per 1 ora poi lavare due volte con la stessa quantità di OS 1X tampone per 10 minuti ciascuno. </li><li> Lascia fiale notte a 4 ° C in tampone 1X OS. </li><li> Fissativo e tutto lava in questa fase vengono smaltiti come rifiuti pericolosi. Tutte le pipette contenenti OsO 4 sono stati sciacquati 3X con olio vegetale. </li></ol> 3. Lavare e disidratazione <ol><li> Il mattino dopo agar blocchi lavare due volte sul ghiaccio con lo stesso volume di cui sopra di acqua fredda per 10 minuti ciascuno utilizzando pipetta Pasteur. </li><li> Lavare in sequenza con lo stesso volume del 25%, 50%, 75%, 95% di etanolo per 10 minuti ciascuno seguito da 2 lavaggi con 100% di etanolo per 10 minuti. Lasciar riposare fino a 4 ° C in 100% di etanolo. </li><li> Tutti i lavaggi in questa fase vengono smaltiti come rifiuti pericolosi. </li></ol> 4. Preparazione di Spurr <ol><li> La mattina seguente (il più presto possibile) pesano le seguenti soluzioni (Scienze EM) in un bicchiere di plastica usa e getta sotto una cappa aspirante per la preparazione del reagente Spurr. Per tutti i lavori successivi usando questo reagente, continuare a utilizzare una cappa aspirante. (ERL 4221 soluzione al 5 grammi) (DER 736 soluzione di 4 grammi) (Soluzione NSA 13 grammi) </li><li> mix di queste soluzioni (lentamente) per 20 minuti nel cofano con un ancoretta </li><li> Aggiungere la soluzione DMAE 0,15 grammi e mescolare 20 minuti come sopra. </li><li> Degas miscela sopra per 1-2 ore nel cappuccio. </li></ol> 5. Infiltrazione di Spurr <ol><li> Appena Spurr è fatta lavare blocchi agar 5 volte con lo stesso volume come sopra del 100% etanolo temperatura ambiente per 10 minuti ciascuno. Dopo il lavaggio etanolo ultima rimuovere l&#39;etanolo con una pipetta Pasteur in modo che l&#39;etanolo residuo copre solo i blocchi di agar. </li><li> Aggiungere circa una pari quantità di reagente Spurr di (circa 0,5 ml) e ruotare fiale sulla ruota per 15 minuti a temperatura ambiente e poi lasciare riposare per 30 minuti. Dopo 30 minuti togliere completamente la soluzione con una pipetta Pasteur, aggiungere Spurr per coprire bloccare agar (circa 1,5 ml), ruotare fiale sulla ruota per 15 minuti a temperatura ambiente e lasciare riposare per 30 minuti. Ripetere la sostituzione della Spurr, la rotazione e l&#39;incubazione altri 3 volte. </li><li> Dopo la finale 30 minuti, rimuovere reagente Spurr e aggiungere nuove Spurr per coprire i blocchi. Lasciare i blocchi agar riposare a temperatura ambiente per 4 ore. </li><li> Sostituire il Spurr e ruotare durante la notte. </li><li> In mattinata sostituire Spurr e lasciare a rotazione fino al giorno successivo. </li><li> Il prossimo Spurr posto mattina in stampi in silicone numerata (Fisher Scientific) solo per coprire il fondo di stampi poi aggiungere blocchi agar per gli stampi. Incubare a 60 ° C per 4 ore </li><li> Dopo 4 ore, in alto fuori gli stampi, con la Spurr più e incubare a 60 ° C durante la notte. </li><li> Se dopo aver rimosso i blocchi dalle muffe i blocchi sono ancora leggermente flessibile, ritorno al stampi e incubare un ulteriore 2 giorni </li></ol> 6. Sezionamento <ol><li> Un Ultracut microtomo Leica S è stato usato per tagliare sezioni 0,5 μ di spessore per la microscopia ottica e 0,1 μ sezioni sottili per EM trasmissione. </li><li> Per la microscopia ottica, come sezioni sono stati tagliati, sono stati collocati su una goccia di dH 2 O, ed essiccato su un blocco di calore ° 52 C. Sezioni secchi sono stati poi colorati con un calo del 1,0% blu di toluidina, borato di sodio 1% per 5-15 secondi. Dopo la colorazione, le sezioni sono state lavate immediatamente sotto un getto d&#39;acqua, secchi, coperti di mezzo di montaggio (KPL), e una scivolata busta chiusa sopra il campione. </li></ol> La rilevanza gamma di misurazione formazione di pattern nel lievito e altri microrganismi sono stati esaminati in precedenza 1. Di particolare nota è la maggiore funzionalità delle colonie microbiche organizzate rispetto alle comunità disorganizzato. Il metodo descritto è una modifica di un metodo per l&#39;incorporamento più grandi colonie specializzate 2, e una breve descrizione delle nostre modifiche sono state pubblicate 3. Un esempio di un microscopio luce di una sezione attraverso il centro di una colonia di S. selvaggio lievito Saccharomyces colonia è mostrata in Figura 1. Aschi, pseudoife e lievito ovoidale sono facilmente distinguibili, e la regione della colonia invadere l&#39;agar sottostante è anche evidente. Un esempio di un microscopio elettronico di un ceppo di lievito di laboratorio (W303 sfondo) è illustrato nella figura 2. L&#39;immagine viene da una regione della colonia che contiene una elevata frequenza di cellule sporigeni. <img src="/files/ftp_upload/2510/2510fig1.jpg" alt="Figura 1" /> Figura 1. Microscopia ottica di una colonia di lieviti selvaggi. Questo lievito (YPS133) è stato isolato recentemente da essudati albero 4. La colonia è stato incubato per 6 giorni in media 5 YNA prima di sezionamento. Le frecce indicano aperto rappresentante aschi, frecce piene indicano rappresentante cellule ovoidali vegetativo, punte di freccia pieni indicano catene di cellule allungate (pseudoife) e aschi. <img src="/files/ftp_upload/2510/2510fig2.jpg" alt="Figura 2" /> Figura 2. Microscopia elettronica di un ceppo di lievito di laboratorio. La colonia di SH1020 (W303 sfondo) è stata incubata per 6 giorni in media YNA prima di sezionamento 3. Immagine mostra una regione della sezione con una frequenza elevata di aschi. Una freccia indica la bi-strato la struttura della parete spore. <img src="/files/ftp_upload/2510/2510fig3.jpg" alt="Figura 3" /> Figura 3. La quantificazione della distribuzione di cellule sporigeni in colonie: A) una griglia contenente 15 rettangoli adiacenti sovrapposti lungo il lato lungo si sovrappone la regione centrale di una immagine di una sezione colonia. La griglia è in scala, mantenendo le proporzioni costante, per ricopra solo la parte superiore e inferiore della colonia. (B &amp; C) la frazione di cellule sporigeni in ogni rettangolo rispetto alle cellule totali in quel rettangolo è determinato mediante ispezione visiva delle immagini da 4 giorni di età (B) e 8 giorni di età (C) colonie. Ad esempio, il rettangolo in alto della colonia (contrassegnata con &quot;1&quot;) corrisponde al lato sinistro del grafico. Media di 4 colonie è mostrato, le barre di errore di visualizzazione std. errore.

<ol>
	<li>Kimmel, A. R., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+symmetries:+regulation+of+Dictyostelium+development+through+chemoattractant+and+morphogen+signal-response.">Breaking symmetries: regulation of Dictyostelium development through chemoattractant and morphogen signal-response.</a> Curr Opin Genet Dev. 14, 540-540 (2004).</li><li>Palkova, Z., Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+within+a+community:+benefit+to+yeast+long-term+survival.">Life within a community: benefit to yeast long-term survival.</a> FEMS Microbiol Rev. 30, 806-806 (2006).</li><li>Shapiro, J., Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significances+of+bacterial+colony+patterns.">The significances of bacterial colony patterns.</a> Bioessays. 17, 597-597 (1995).</li><li>Shapiro, J., Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinking+about+bacterial+populations+as+multicellular+organisms.">Thinking about bacterial populations as multicellular organisms.</a> Annu Rev Microbiol. 52, 81-81 (1998).</li><li>Engelberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+stalk-like+structures+in+Saccharomyces+cerevisiae.">Multicellular stalk-like structures in Saccharomyces cerevisiae.</a> J Bacteriol. 180, 3992-3992 (1998).</li><li>Scherz, R., Shinder, V., Engelberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+analysis+of+Saccharomyces+cerevisiae+stalk-like+structures+reveals+spatial+organization+and+cell+specialization.">Anatomical analysis of Saccharomyces cerevisiae stalk-like structures reveals spatial organization and cell specialization.</a> J Bacteriol. 183, 5402-5402 (2001).</li><li>Piccirillo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rim101p/PacC+pathway+and+alkaline+pH+regulate+pattern+formation+in+yeast+colonies.">The Rim101p/PacC pathway and alkaline pH regulate pattern formation in yeast colonies.</a> Genetics. 184, 707-707 (2010).</li><li>Sniegowski, P. D., Dombrowski, P. G., Fingerman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccharomyces+cerevisiae+and+Saccharomyces+paradoxus+coexist+in+a+natural+woodland+site+in+North+America+and+display+different+levels+of+reproductive+isolation+from+European+conspecifics.">Saccharomyces cerevisiae and Saccharomyces paradoxus coexist in a natural woodland site in North America and display different levels of reproductive isolation from European conspecifics.</a> FEMS Yeast Res. 1, 299-299 (2002).</li><li>Piccirillo, S., Honigberg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporulation+patterning+and+invasive+growth+in+wild+and+domesticated+yeast+colonies.">Sporulation patterning and invasive growth in wild and domesticated yeast colonies.</a> Res Microbiol. 161, 390-390 (2002).</li><li>Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+developing+multicellular+yeast+colony:+spatio-temporal+expression+of+Ato1p+ammonium+exporter.">Architecture of developing multicellular yeast colony: spatio-temporal expression of Ato1p ammonium exporter.</a> Environ Microbiol. , (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

Il metodo presentato rivela la struttura interna delle colonie. Poiché il metodo è efficace nel determinare i modelli di tipi di cellule in una gamma di S. ceppi di Saccharomyces con diverse morfologie di colonia, e anche in una specie legate S. paradoxus 5, il metodo è anche probabile che il lavoro su una vasta gamma di funghi e altri microrganismi. Un passo fondamentale per il successo del metodo è quello di garantire che l&#39;intera colonia, compresa la p...

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<td>Osmium tetroxide</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 19152</td>
<td>&nbsp;</td>
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<td>Silicone embedding molds</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>NC 9975029</td>
<td>&nbsp;</td>
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<td>Cycloaliphatic epoxide resin</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 15004</td>
<td>ERL 4221</td>
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<tr>
<td>Epoxy resin</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 13000</td>
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<td>Nonenyl Succinic Anhydride</td>
<td>&nbsp;</td>
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yeast colony embedding method

Patterning di diversi tipi di cellule in embrioni è un meccanismo chiave dello sviluppo metazoi. Comunità di microrganismi, come colonie e biofilm anche visualizzare modelli di tipi di cellule. Per esempio, nel lievito S. cerevisiae, le cellule e le cellule pseudohyphal sporigeni non sono distribuite uniformemente in colonie. L&#39;importanza funzionale di patterning e dei meccanismi molecolari che sono alla base di questi modelli sono ancora poco conosciuti. Una sfida rispetto a indagare i modelli di tipi di cellule in colonie fungine è che a differenza dei tessuti metazoi, le cellule in colonie sono relativamente debolmente attaccati l&#39;uno all&#39;altro. In particolare, le colonie fungine non contengono lo stesso livello ampia di matrice extracellulare trovano nella maggior parte dei tessuti. Qui riportiamo un metodo per l&#39;integrazione e sezionamento colonie di lievito che rivela i modelli interni di tipi di cellule in queste colonie. Il metodo può essere utilizzato per preparare sezioni spesse (0,5 μ) utile per la microscopia ottica e di sezioni sottili (0,1 μ) adatto per la microscopia elettronica a trasmissione. Cellule aschi e pseudohyphal può essere facilmente distinto da cellule di lievito ovoidale al microscopio ottico, mentre la struttura interna di queste cellule possono essere visualizzati da EM. Il metodo si basa su colonie circostanti con agar, infiltrandosi con media Spurr, e poi sezionamento. Colonie con un diametro nel range di 1-2 mm sono adatte per questo protocollo. Oltre a visualizzare l&#39;interno delle colonie, il metodo consente la visualizzazione della regione della colonia che invade l&#39;agar sottostante.

Watch this Scientific Journal Video about Yeast Colony Embedding Method at JoVE.com

Yeast Colony Embedding Method

Un metodo per l&#39;integrazione di colonie di lievito che permette di sezionamento per la microscopia ottica ed elettronica. Questo protocollo consente di determinare la distribuzione delle cellule sporigeni e cellule pseudohyphal all&#39;interno di colonie disposizione un nuovo strumento per comprendere l&#39;organizzazione di tipi di cellule all&#39;interno di una comunità fungine.

Lievito Colony metodo di inserimento

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

Research

JoVE Journal

Biology

11.4K Views.  University of Missouri - Kansas City. Un metodo per l'integrazione di colonie di lievito che permette di sezionamento per la microscopia ottica ed elettronica. Questo protocollo consente di determinare la distribuzione delle cellule sporigeni e cellule pseudohyphal all'interno di colonie disposizione un nuovo strumento per comprendere l'organizzazione di tipi di cellule all'interno di una comunità fungine.

Video: Yeast Colony Embedding Method

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 ...

Ricerca

Biologia

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Purificazione dei mitocondri da cellule di lievito

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

High-throughput iperespressione schermo lievito plasmidi

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Schermi competitivo genomica delle Biblioteche di lievito con codice a barre

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

Un dosaggio quantitativo di insulina che esprimono formante colonia Progenitori

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Purificazione cromatografica dei ribosomi di lievito Highly Active

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Microbes typically live in communities. The spatial organization of cells within a community is believed to impact the survival and function of the community1. Optical sectioning techniques, including confocal and two-photon microscopy, have proven useful for observing spatial organization of bacterial and archaeal communities2,3. A combination of confocal imaging and physical sectioning of yeast colonies has revealed internal organization of cells4. However, direct optical sectioning using confocal or two-photon microscopy has been only able to reach a few cell layers deep into yeast colonies. This limitation is likely because of strong scattering of light from yeast cells4.
Here, we present a method based on fixing and cryosectioning to obtain spatial distribution of fluorescent cells within Saccharomyces cerevisiae communities. We use methanol as the fixative agent to preserve the spatial distribution of cells. Fixed communities are infiltrated with OCT compound, frozen, and cryosectioned in a cryostat. Fluorescence imaging of the sections reveals the internal organization of fluorescent cells within the community.
Examples of yeast communities consisting of strains expressing red and green fluorescent proteins demonstrate the potentials of the cryosectioning method to reveal the spatial distribution of fluorescent cells as well as that of gene expression within yeast colonies2,3. Even though our focus has been on Saccharomyces cerevisiae communities, the same method can potentially be applied to examine other microbial communities.

We present a protocol for freezing and cryosectioning yeast communities to observe internal patterns of fluorescent cells. The method relies on methanol-fixing and OCT-embedding to preserve the spatial distribution of cells without inactivating fluorescent proteins within a community.

Criosezionamento Comunità di lievito per l&#39;esame di pattern di fluorescenza

Microbes typically live  in communities. The spatial organization  of cells within a community is believed to impact the survival and function of the  ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

Il saggio Formazione morbida Agar Colony

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

Diffondere superfici e Immunostaining di cromosomi del lievito

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

Microscopia di lievito di fissione del ciclo di vita sessuale

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...