La investigación fue financiada por el NIH 1R15GM094770.

1. Colonia El aislamiento y la fijación <ol><li> Incubar 300 colonias en un medio de agar durante el tiempo indicado (una colonia aislada debe ser de 1-2 mm de diámetro). </li><li> Retire la colonia (boca arriba) y medio subyacente utilizando una espátula. </li><li> Coloque varias gotas de 2% de agar 42 ° C en un portaobjetos con 1 mL Pipetman punta y de inmediato el lugar de colonias en el rostro de agar antes de que se solidifica. </li><li> Coloque varias gotas de 2% de agar 42 ° C en la colonia y dejar que se solidifique. </li><li> Use guantes para todos los pasos en lo que resta de este protocolo </li><li> Bloque de corte con cuchilla de afeitar y colocarlos en un 3,5 ml de borosilicato con tapón de rosca vial que contiene 2% de paraformaldehído / 2% fijador de glutaraldehído durante 7 días a 4 ° C. </li></ol> 2. Se lava y tratamiento de osmio <ol><li> Después de aproximadamente una semana, lavado de bloques de agar en el hielo bajo campana para sustancias químicas mediante la incubación de dos veces durante 15 minutos con cacodilato de sodio 0,15 M suficiente (pH 7,2) para cubrir por completo la colonia (aproximadamente 1,5 ml) y luego dos veces más durante 5 minutos con el mismo volumen 1X buffer de SO (100 mM KH 2 PO 4 10 mM MgCl 2, pH 6,0). </li><li> Añadir el mismo volumen de 1% de OsO 4 [2 ml OsO 4 (2%) + 2 ml de 2x OS Buffer] para viales (en hielo) para cubrir los bloques de agar en campana para sustancias químicas de 1 hora y luego lavar dos veces con la misma cantidad 1X buffer de SO durante 10 minutos cada uno. </li><li> Deja viales durante la noche a 4 ° C en 1X buffer del sistema operativo. </li><li> Fijador y todos los lavados en este paso se eliminan como residuos peligrosos. Todas las pipetas contienen OsO4 se lavaron 3X con aceite vegetal. </li></ol> 3. Lavado y deshidratación <ol><li> A la mañana siguiente bloques de agar lavado dos veces en el hielo con el mismo volumen que el anterior de agua fría durante 10 minutos cada uno con una pipeta Pasteur. </li><li> Lavar sucesivamente con el mismo volumen de 25%, 50%, 75%, 95% de etanol durante 10 minutos cada uno seguido por dos lavados con etanol al 100% durante 10 minutos. Dejar toda la noche a 4 ° C en el 100% de etanol. </li><li> Todos los lavados en este paso se eliminan como residuos peligrosos. </li></ol> 4. Preparación de Spurr <ol><li> A la mañana siguiente (lo más temprano posible) pesan las siguientes soluciones (Ciencias de la EM) en un vaso de plástico desechable en una campana de extracción para preparar reactivos de Spurr. Para todos los trabajos posteriores de este método, siga utilizando una campana de humos. (ERL 4221 solución de 5 gramos) (DER 736 solución de 4 gramos) (Solución NSA 13 gramos) </li><li> mezcla de estas soluciones (lentamente) durante 20 minutos en campana es suficiente utilizar una barra de agitación </li><li> Añadir la solución de DMAE 0,15 gramos y revuelva 20 minutos anteriores. </li><li> Degas por encima de la mezcla durante 1-2 horas en la campana. </li></ol> 5. La infiltración de Spurr <ol><li> Tan pronto como Spurr se lave bloques de agar 5 veces con un volumen igual al anterior de 100% de etanol temperatura ambiente durante 10 minutos cada uno. Después del lavado de etanol última separar el etanol con una pipeta Pasteur para que el etanol restante apenas cubre los bloques de agar. </li><li> Añadir aproximadamente la misma cantidad de reactivo de Spurr (aproximadamente 0,5 ml) y girar viales en la rueda durante 15 minutos a temperatura ambiente y se deja reposar durante 30 minutos. Después de 30 minutos, retire la solución completa con una pipeta Pasteur, añadir Spurr de cubierta del bloque de agar (aproximadamente 1,5 ml), gire viales en la rueda durante 15 minutos a temperatura ambiente y dejar reposar durante 30 minutos. Repita el reemplazo de Spurr, rotación, y la incubación de 3 veces más. </li><li> Después de los últimos 30 minutos, retire los reactivos de Spurr y añadir nuevas Spurr para cubrir los bloques. Permita que los bloques de agar para estar a temperatura ambiente durante 4 horas. </li><li> Vuelva a colocar la de Spurr y rotar durante la noche. </li><li> En la mañana reemplazar Spurr y dejar girando hasta el día siguiente. </li><li> El Spurr mañana siguiente lugar en moldes de silicona numeradas (Fisher Scientific) sólo para cubrir el fondo de los moldes a continuación, añadir bloques de agar a los moldes. Se incuba a 60 ° C durante 4 horas </li><li> Después de 4 horas, parte superior de los moldes, con los más Spurr y se incuba a 60 ° C durante la noche. </li><li> Si después de retirar los bloques de los moldes de los bloques son todavía un poco flexibles, volver a los moldes y se incuba un adicional de 2 días </li></ol> 6. Seccionamiento <ol><li> Un microtomo Ultracut Leica S fue utilizado para cortar secciones de 0,5 μ de espesor para microscopía de luz y 0,1 μ secciones delgadas de EM de transmisión. </li><li> Para microscopía de luz, ya que se cortaron secciones, que fueron colocados en una gota de dH 2 O, y se seca en un 52 ° C del bloque de calor. Secciones de secado fueron teñidas con una caída de 1,0% de azul de toluidina, el 1% borato de sodio de 5.15 segundos. Después de la tinción, las secciones se lavaron inmediatamente bajo un chorro de agua, se seca, cubierta en medio de montaje (KPL) y una hoja de cubierta sellada en la muestra. </li></ol> La amplia relevancia de la medición de la formación de patrones en la levadura y otros microorganismos han sido revisados ​​previamente 1. De particular interés es el aumento de la funcionalidad de las colonias microbianas organizadas en relación con las comunidades desorganizadas. El método descrito es una modificación de un método para la incorporación de grandes colonias especializados 2, y una breve descripción de nuestras modificaciones se han publicado 3. Un ejemplo de una micrografía de luz de una sección a través del centro de una colonia salvaje de S. colonia de levadura Saccharomyces se muestra en la Figura 1. Ascos, pseudohifas y levaduras ovoides son fácilmente distinguibles, y en la región de la colonia invadir el agar subyacente es también evidente. Un ejemplo de una micrografía electrónica de una cepa de laboratorio de la levadura (W303 de fondo) se muestra en la Figura 2. La imagen es de una región de la colonia que contiene una alta frecuencia de células esporuladas. <img src="/files/ftp_upload/2510/2510fig1.jpg" alt="Figura 1" /> Figura 1. Microscopía de luz de una colonia de hongos silvestres. Esta levadura (YPS133) fue aislada recientemente de 4 exudados de árboles. La colonia se incubó durante 6 días en medio de YNA 5 antes de cortar. Flechas abiertas indican representante ascos, lleno de flechas indican las células vegetativas representante ovoide, puntas de flecha indican lleno de cadenas de células alargadas (pseudohifas) y ascos. <img src="/files/ftp_upload/2510/2510fig2.jpg" alt="Figura 2" /> Figura 2. Microscopia electrónica de una cepa de levadura de laboratorio. La colonia de SH1020 (W303 de fondo) se incubó durante 6 días en el medio YNA antes de seccionar 3. La imagen muestra una región de la sección con una alta frecuencia de ascos. Una flecha indica la estructura de dos capas de la pared de la espora. <img src="/files/ftp_upload/2510/2510fig3.jpg" alt="Figura 3" /> Figura 3. La cuantificación de la distribución de células esporuladas en las colonias de: A) una cuadrícula que contiene 15 rectángulos contiguos apilados a lo largo de su lado más largo se superpone a la región central de la imagen de una sección de la colonia. La red es reducido, manteniendo sus proporciones constantes, que sólo cubre la parte superior e inferior de la colonia. (B &amp; C) La fracción de células esporuladas en cada rectángulo en relación con el total de células en el rectángulo que se determina por inspección visual de las imágenes de los viejos de 4 días (B) y 8 días de las antiguas colonias (C). Por ejemplo, el rectángulo en la parte superior de la colonia (&quot;1&quot;) corresponde a la parte izquierda de la gráfica. La media de cuatro colonias se muestra, las barras de error de visualización de la std. error.

<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>

El método presentado revela las estructuras interiores de las colonias. Debido a que el método es eficaz en la determinación de patrones de tipos de células en una variedad de S. cerevisiae con morfologías diferentes colonias, y también en una especie relacionada S. paradoxus 5, el método también es probable que trabajar en una amplia gama de hongos y otros microorganismos. Un paso fundamental para el éxito del método es asegurar que toda la colonia, incluyendo ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<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>
<tr>
<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>
</tr>
<tr>
<td>2-Dimethyl aminoethanol	</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 13300</td>
<td>DMAE</td>
</tr>
<tr>
<td>Mounting media</td>
<td>&nbsp;</td>
<td>KPL</td>
<td>71-00-16</td>
<td>&nbsp;</td>
<tr>
<td>Rotating wheel</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>Pelco 1055</td>
<td>&nbsp;</td>
<tr>
<td>Microtome</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>Ultracut S</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

yeast colony embedding method

Patrón de los diferentes tipos de células en embriones es un mecanismo clave en el desarrollo de los metazoos. Comunidades de microorganismos, tales como colonias y biofilms también muestran patrones de tipos de células. Por ejemplo, en el de levadura de S. cerevisiae, células y las células esporuladas pseudohyphal no están distribuidas uniformemente en las colonias. La importancia funcional de los patrones y los mecanismos moleculares que subyacen a estos modelos son aún poco conocidos. Uno de los retos con respecto a la investigación de patrones de tipos de células en las colonias de hongos es que a diferencia del tejido metazoos, las células en las colonias son relativamente débilmente unidas entre sí. En particular, las colonias de hongos no contienen el mismo nivel amplio de la matriz extracelular en la mayoría de los tejidos. Aquí nos informe sobre un método para la inclusión y corte de colonias de levaduras que revela los patrones interior de tipos de células en estas colonias. El método puede ser usado para preparar secciones gruesas (0,5 μ) es útil para microscopía óptica y secciones delgadas (0,1 μ) adecuada para la microscopía electrónica de transmisión. Células ascos y pseudohyphal se pueden distinguir fácilmente de las células de levadura ovoide por microscopía de luz, mientras que la estructura interior de estas células puede ser visualizado por EM. El método se basa en los alrededores de las colonias con agar, infiltrándose en ellos con el medio de Spurr, y luego el corte. Las colonias con un diámetro en el rango de 1-2 mm son adecuados para este protocolo. Además de visualizar el interior de las colonias, el método permite la visualización de la región de la colonia que invade el agar subyacente.

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

Yeast Colony Embedding Method

Un método para la incorporación de colonias de levadura que permite el corte de luz y microscopía electrónica. Este protocolo permite la determinación de la distribución de células y las células esporuladas pseudohyphal dentro de las colonias de proporcionar una nueva herramienta en la comprensión de la organización de los tipos de células dentro de una comunidad de hongos.

Colonia de levaduras Método de anidamiento

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 vistas.  University of Missouri - Kansas City. Un método para la incorporación de colonias de levadura que permite el corte de luz y microscopía electrónica. Este protocolo permite la determinación de la distribución de células y las células esporuladas pseudohyphal dentro de las colonias de proporcionar una nueva herramienta en la comprensión de la organización de los tipos de células dentro de una comunidad de hongos.

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.

Medición de Span replicativa vida en la levadura en ciernes

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

Investigación

Biología

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.

Purificación de las mitocondrias de las células de levadura

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.

La levadura de alto rendimiento de la pantalla sobreexpresión plásmido

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.

Pantallas competitiva Genómica de Bibliotecas de levadura con código de barras

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 análisis cuantitativo de la insulina que expresan la formación de colonias de progenitores

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.

Purificación por cromatografía de ribosomas de levadura de gran actividad

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.

Cryosectioning Comunidades levadura para examinar los patrones de fluorescencia

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.

El ensayo de formación de colonias en agar blando

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.

Difusión de superficies y inmunotinción de cromosomas de la levadura

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.

Microscopía de la levadura de fisión del ciclo de vida sexual

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