La recherche a été financée par le NIH 1R15GM094770.

1. Isolement des colonies et de fixation <ol><li> Incuber 300 colonies sur milieu gélosé pendant le temps indiqué (une colonie isolée devrait être de 1-2 mm de diamètre). </li><li> Retirez la colonie (face) et moyennes sous-jacentes à l&#39;aide d&#39;une spatule étroite. </li><li> Placez quelques gouttes d&#39;agar à 2% 42 ° C sur une lame de microscope en utilisant 1 ml Pipetman pointe et la colonie place immédiatement sur le visage d&#39;agar-vous avant qu&#39;il se solidifie. </li><li> Placez quelques gouttes d&#39;agar à 2% 42 ° C sur la colonie et laisser se solidifier. </li><li> Portez des gants pour toutes les étapes jusqu&#39;à la fin de ce protocole </li><li> Bloc de Trim avec lame de rasoir et les placer dans un borosilicate 3,5 mL à bouchon à vis flacon contenant 2% de paraformaldéhyde / fixateur glutaraldéhyde à 2% pendant 7 jours à 4 ° C. </li></ol> 2. Lavages et traitements Osmium <ol><li> Après environ 1 semaine, se laver les blocs de glace sous le agar chimiques hotte en incubant à deux reprises pendant 15 minutes avec assez de cacodylate de sodium 0,15 M (pH 7,2) pour couvrir complètement la colonie (environ 1,5 mL) puis deux fois plus pendant 5 minutes avec le même volume de 1X OS tampon (100 mM KH 2 PO 4 10 mM MgCl 2, pH 6,0). </li><li> Ajouter le même volume de 1% OsO 4 [2 mL OsO 4 (2%) + 2 ml de tampon 2x OS] pour flacons (sur glace) pour couvrir les blocs de gélose sous hotte pendant 1 heure puis laver deux fois avec la même quantité de 1X OS tampon pour 10 minutes chacune. </li><li> Laisser les flacons nuit à 4 ° C en 1X OS tampon. </li><li> Fixateur et tous les lavages dans cette étape sont éliminés comme des déchets dangereux. Toutes les pipettes contenant OsO 4 ont été rincées 3X avec l&#39;huile végétale. </li></ol> 3. Lavez et déshydratation <ol><li> Le lendemain matin, lavez les blocs de gélose deux fois sur la glace avec le même volume que ci-dessus de l&#39;eau froide pendant 10 minutes chacun utilisant une pipette Pasteur. </li><li> Laver séquentiellement avec le même volume de 25%, 50%, 75%, 95% d&#39;éthanol pour 10 minutes chacune suivie de 2 lavages avec de l&#39;éthanol à 100% pendant 10 minutes. Laisser une nuit à 4 ° C dans de l&#39;éthanol à 100%. </li><li> Tous les lavages dans cette étape sont éliminés comme des déchets dangereux. </li></ol> 4. Préparation de Spurr <ol><li> Le matin suivant (le plus tôt possible) soupeser les solutions suivantes (EM Sciences) dans un bécher en plastique jetables sous une hotte à préparer le réactif de Spurr. Pour tous les travaux ultérieurs utilisant ce réactif, continuer à utiliser une hotte. (ERL 4221 solution à 5 grammes) (DER 736 solution de 4 grammes) (Solution NSA 13 grammes) </li><li> mélange de ces solutions (lentement) pendant 20 minutes dans le capot à l&#39;aide d&#39;un barreau </li><li> Ajouter la solution de 0,15 g de DMAE et remuer 20 minutes comme ci-dessus. </li><li> Degas mélange ci-dessus pendant 1-2 heures dans le capot. </li></ol> 5. Spurr Infiltration <ol><li> Dès que Spurr est fait laver les blocs de gélose à 5 reprises avec le même volume que ci-dessus de 100 d&#39;éthanol temp% ambiante pendant 10 minutes chacun. Après le lavage à l&#39;éthanol dernière éliminer l&#39;éthanol à l&#39;aide d&#39;une pipette Pasteur afin que l&#39;éthanol restant couvre simplement les blocs de gélose. </li><li> Ajouter environ un montant égal de réactif de Spurr (environ 0,5 ml) et faire tourner la roue de flacons pendant 15 minutes à température ambiante, puis laisser reposer pendant 30 minutes. Après 30 minutes retirer la solution complète à l&#39;aide d&#39;une pipette Pasteur, ajouter Spurr pour couvrir bloc de gélose (environ 1,5 mL), tourner sur la roue flacons pendant 15 minutes à température ambiante et laisser reposer pendant 30 minutes. Répétez le remplacement du Spurr, la rotation et d&#39;incubation encore 3 fois. </li><li> Après les 30 dernières minutes, retirez réactif Spurr et ajouter frais de Spurr pour couvrir les blocs. Autoriser les blocs de gélose, pour s&#39;établir à température ambiante pendant 4 heures. </li><li> Remplacer les années Spurr et tourner toute la nuit. </li><li> Dans la matinée, remplacez Spurr et laisser tourner jusqu&#39;à ce que le jour suivant. </li><li> Le lendemain matin Spurr lieu dans des moules en silicone numérotés (Fisher Scientific), juste pour couvrir le fond des moules, puis ajouter des blocs de gélose pour les moules. Incuber à 60 ° C pendant 4 heures </li><li> Après 4 heures, couronner les moules, avec plus de Spurr et incuber à 60 ° C pendant la nuit. </li><li> Si après avoir enlevé les blocs à partir de moules, les blocs sont encore légèrement flexible, le retour aux moules et laisser incuber 2 jours supplémentaires </li></ol> 6. Sectionnement <ol><li> Un microtome Ultracut Leica S a été utilisée pour couper des sections 0.5 μ d&#39;épaisseur pour la microscopie optique et 0,1 μ lames minces pour EM transmission. </li><li> Pour la microscopie optique, que les sections ont été coupés, ils ont été placés sur une goutte de dH 2 O, et séché sur un bloc de 52 ° C chaleur. Sections séchées ont ensuite été colorées avec une baisse de 1,0% de bleu de toluidine, borate de sodium à 1% pendant 5-15 secondes. Après coloration, les sections ont été immédiatement lavées sous un jet d&#39;eau, séché, couvert par les médias de montage (KPL), et une lamelle scellés sur l&#39;échantillon. </li></ol> La grande pertinence de mesurer la formation de modèles dans la levure et autres micro-organismes ont été examinées précédemment 1. On notera en particulier la fonctionnalité accrue des colonies microbiennes organisées par rapport à des communautés désorganisé. La méthode décrite est une modification d&#39;une méthode pour intégrer de plus grandes colonies spécialisée 2, et une courte description de nos modifications a été publiée 3. Un exemple de la microscopie optique d&#39;une section à travers le centre d&#39;une colonie de sauvages S. colonie de levures cerevisiae est montré dans la figure 1. Asques, pseudohyphae et la levure ovoïde sont facilement identifiables, et dans la région de la colonie envahir la gélose sous-jacente est également manifeste. Un exemple d&#39;un microscope électronique d&#39;une souche de laboratoire de la levure (W303 de fond) est montré dans la figure 2. L&#39;image est d&#39;une région de la colonie contenant une fréquence élevée de cellules sporulées. <img src="/files/ftp_upload/2510/2510fig1.jpg" alt="Figure 1" /> Figure 1. Microscopie optique d&#39;une colonie de levures sauvages. Cette levure (YPS133) a été isolé récemment de 4 exsudats d&#39;arbres. La colonie a été incubée pendant 6 jours sur YNA moyenne 5 avant la coupe. Flèches indiquent Ouvrez représentant asques, les flèches indiquent remplis représentant ovoïde cellules végétatives, pointes de flèches pleins indiquent des chaînes de cellules allongées (pseudohyphes) et asques. <img src="/files/ftp_upload/2510/2510fig2.jpg" alt="Figure 2" /> Figure 2. Microscopie électronique d&#39;une souche de levure de laboratoire. La colonie de SH1020 (W303 fond) a été incubée pendant 6 jours sur milieu YNA avant la coupe 3. L&#39;image montre une région de la section avec une fréquence élevée des asques. Une flèche indique la structure bi-couche du mur de spores. <img src="/files/ftp_upload/2510/2510fig3.jpg" alt="Figure 3" /> Figure 3. Quantification de la distribution des cellules sporulées dans les colonies: A) une grille contenant 15 rectangles adjacents empilés le long de leur côté le plus long est superposée à la région centrale de l&#39;image d&#39;une section colonie. La grille est mise à l&#39;échelle, tout en gardant ses proportions constantes, pour ne couvre que le haut et le bas de la colonie. (B &amp; C) La fraction de cellules sporulées dans chaque rectangle par rapport aux cellules totales dans ce rectangle est déterminée par inspection visuelle des images à partir de 4 jours anciens (B) et de 8 jours anciens (C) des colonies. Par exemple, le rectangle en haut de la colonie («1») correspond à la partie gauche du graphique. Moyenne de 4 colonies est montré; les barres d&#39;erreur d&#39;affichage du MST. erreur.

<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>

La méthode présentée révèle les structures internes des colonies. Parce que la méthode est efficace pour déterminer les schémas de types de cellule dans une plage de S. souches cerevisiae avec des morphologies différentes colonies, et aussi dans une espèce apparentée S. paradoxus 5, la méthode est également susceptible de travailler sur un large éventail de champignons et autres micro-organismes. Une étape cruciale pour le succès de la méthode es...

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<td>Osmium tetroxide</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 19152</td>
<td>&nbsp;</td>
<tr>
<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>
</tr>
<tr>
<td>Epoxy resin</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 13000</td>
<td>DER 736</td>
</tr>
<tr>
<td>Nonenyl Succinic Anhydride</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 19050</td>
<td>NSA</td>
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<td>2-Dimethyl aminoethanol	</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Ted Pella</td>
<td>Pelco 1055</td>
<td>&nbsp;</td>
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yeast colony embedding method

Modélisation des différents types de cellules dans des embryons est un mécanisme clé dans le développement des métazoaires. Communautés de microorganismes, comme les colonies et les biofilms également afficher les modèles de types cellulaires. Par exemple, dans la levure S. cerevisiae, les cellules et les cellules sporulées pseudohyphal ne sont pas réparties uniformément dans les colonies. L&#39;importance fonctionnelle de la structuration et les mécanismes moléculaires qui sous-tendent ces tendances sont encore mal compris. Un défi à l&#39;égard de schémas de enquêter types de cellules dans les colonies fongiques, c&#39;est que contrairement au tissu métazoaires, les cellules en colonies sont relativement faiblement attaché à un autre. En particulier, colonies de champignons ne contiennent pas le même niveau complète de la matrice extracellulaire dans la plupart des tissus. Nous rapportons ici une méthode pour intégrer et de sectionnement des colonies de levures qui révèle les habitudes intérieures de types de cellules dans ces colonies. La méthode peut être utilisée pour préparer des coupes épaisses (0,5 μ) utiles pour la microscopie optique et lames minces (0,1 μ) adapté à la microscopie électronique à transmission. Cellules asques et pseudohyphal peuvent être facilement distingués des cellules de levure ovoïde par microscopie optique, tandis que la structure intérieure de ces cellules peuvent être visualisées par EM. La méthode est basée sur les colonies environnantes avec de l&#39;agar, les infiltrer avec des moyennes de Spurr, puis sectionner. Colonies avec un diamètre de l&#39;ordre de 1-2 mm sont adaptés à ce protocole. En plus de visualiser l&#39;intérieur des colonies, la méthode permet la visualisation de la région de la colonie qui envahit la gélose sous-jacente.

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

Yeast Colony Embedding Method

Une méthode pour l&#39;intégration des colonies de levures permettant la coupe pour la microscopie optique et électronique. Ce protocole permet la détermination de la distribution de cellules et les cellules sporulées pseudohyphal au sein des colonies fournissant un nouvel outil pour comprendre l&#39;organisation de types de cellules dans une communauté fongiques.

Méthode d&#39;insertion de levure Colony

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 vues.  University of Missouri - Kansas City. Une méthode pour l'intégration des colonies de levures permettant la coupe pour la microscopie optique et électronique. Ce protocole permet la détermination de la distribution de cellules et les cellules sporulées pseudohyphal au sein des colonies fournissant un nouvel outil pour comprendre l'organisation de types de cellules dans une communauté fongiques.

Vidéo: 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.

Mesure Life Span réplicative dans la levure bourgeonnante

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

Recherche

Biologie

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.

Purification des mitochondries de cellules de levure

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.

Haut débit écran surexpression plasmide de levure

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.

Écrans concurrentiel des bibliothèques génomiques de levure Barcoded

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 dosage quantitatif de l&#39;insuline exprimer formant colonie Progéniteurs

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.

Purification chromatographique des ribosomes de levure hautement 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.

Découpe au cryostat Communautés de levure pour examen des caractéristiques de fluorescence

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.

La formation de dosage souple 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.

Répandre surface et Immunocoloration des chromosomes de levure

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.

Microscopie de levure Fission Lifecycle sexuelle

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