Queremos agradecer a todos los miembros de nuestro laboratorio y el Centro para la Biología de Sistema Integrado de Envejecimiento y Nutrición (CISBAN) para el apoyo y útiles debates. Este estudio fue apoyado por las Ciencias Biológicas y la Biotecnología Research Council (BBSRC) (BB/C008200/1) y el Wellcome Trust (075294, 093088).

<ol>
	<li>Addinall, S. G., Holstein, E., Lawless, C., Yu, M., Chapman, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Fitness+Analysis+shows+that+NMD+proteins+and+many+other+protein+complexes+suppress+or+enhance+distinct+telomere+cap+defects.">Quantitative Fitness Analysis shows that NMD proteins and many other protein complexes suppress or enhance distinct telomere cap defects.</a> PLoS Genetics. 7 (4), e1001362 (2011).</li><li>Addinall, S. G., Downey, M., Yu, M., Zubko, M. K., Dewar, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genomewide+suppressor+and+enhancer+analysis+of+cdc13-1+reveals+varied+cellular+processes+influencing+telomere+capping+in+Saccharomyces+cerevisiae.">A genomewide suppressor and enhancer analysis of cdc13-1 reveals varied cellular processes influencing telomere capping in Saccharomyces cerevisiae.</a> Genetics. 180, 2251-2266 (2008).</li><li>Lawless, C., Wilkinson, D., Young, A., Addinall, S., Lydall, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonyzer:+automated+quantification+of+micro-organism+growth+characteristics+on+solid+agar.">Colonyzer: automated quantification of micro-organism growth characteristics on solid agar.</a> BMC Bioinformatics. 11, 287-28 (2010).</li><li>Tong, A. H., Evangelista, M., Parsons, A. B., Xu, H., Bader, G. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+genetic+analysis+with+ordered+arrays+of+yeast+deletion+mutants.">Systematic genetic analysis with ordered arrays of yeast deletion mutants.</a> Science. 294, 2364-2368 (2001).</li><li>Costanzo, M., Baryshnikova, A., Bellay, J., Kim, Y., Spear, E. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+landscape+of+a+cell.">The genetic landscape of a cell.</a> Science. 327, 425-431 (2010).</li><li>Blomberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+growth+rate+in+high-throughput+growth+phenotyping.">Measuring growth rate in high-throughput growth phenotyping.</a> Curr. Opin. Biotechnol. 22 (1), 94-102 (2011).</li><li>Maringele, L., Lydall, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EXO1-dependent+single-stranded+DNA+at+telomeres+activates+subsets+of+DNA+damage+and+spindle+checkpoint+pathways+in+budding+yeast+yku70&#916;mutants.">EXO1-dependent single-stranded DNA at telomeres activates subsets of DNA damage and spindle checkpoint pathways in budding yeast yku70&#916;mutants.</a> Genes and Dev. 16 (15), 1919-1933 (2002).</li><li>Warringer, J., Blomberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+screening+in+environmental+arrays+allows+analysis+of+quantitative+phenotypic+profiles+in+Saccharomyces+cerevisiae.">Automated screening in environmental arrays allows analysis of quantitative phenotypic profiles in Saccharomyces cerevisiae.</a> Yeast. 20, 53-67 (2003).</li><li>Hillenmeyer, M., Fung, E., Wildenhain, J., Pierce, S., Hoon, S., Lee, W., Proctor, M., St. Onge, R., Tyers, M., Koller, D., Altman, R., Davis, R., Nislow, C., Giaever, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemical+genomic+portrait+of+yeast:+uncovering+a+phenotype+for+all+genes.">The chemical genomic portrait of yeast: uncovering a phenotype for all genes.</a> Science. 320 (5874), 362-365 (2008).</li><li>Smith, A. M., Durbic, T., Oh, J., Urbanus, M., Proctor, M., Heisler, L. E., Giaever, G., Nislow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competitive+Genomic+Screens+of+Barcoded+Yeast+Libraries.">Competitive Genomic Screens of Barcoded Yeast Libraries.</a> J. Vis. Exp. (54), e2864 (2011).</li><li>Lederberg, J., Lederberg, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replica+plating+and+indirect+selection+of+bacterial+mutants.">Replica plating and indirect selection of bacterial mutants.</a> J. Bacteriol. 63, 399-406 (1952).</li><li>Queller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+ecology:+The+social+side+of+wild+yeast.">Behavioural ecology: The social side of wild yeast.</a> Nature. 456, 589-590 (2008).</li></ol>

No hay conflictos de interés declarado.

QFA es en muchos sentidos, un descendiente directo de los tres establecidos a escala de banco técnicas microbiológicas: cultura dilución, pruebas in situ de la serie 9, la determinación de la curva de crecimiento en cultivos líquidos aireados y las planchas del 9 de réplica 13. Estos tres métodos se resumen y se comparan con QFA y otras técnicas de alto rendimiento en la Tabla 2. Considerando que la dilución, pruebas in situ de la serie definirá la aptitud de una cepa como su capacidad de formar colonias en una serie de culturas cultivadas a partir de una serie de diluciones del inóculo, QFA gimnasio cepa medidas por parte de las observaciones repetidas de una misma cultura para construir una curva de crecimiento. Cuantificación de la aptitud de los cultivos individuales permite muchas cepas más a ser examinada simultáneamente, bajo condiciones idénticas. Replicar las matrices de las culturas permite repetir la prueba de las colecciones de la cepa, el más útil cuando las pruebas de diferencias de aptitud de cultivo en diferentes ambientes o las bases genéticas. El protocolo presentado QFAaquí utiliza equipos robóticos costosas de duplicar, inocular, incubar y las placas de agar de imagen, adecuada para observar el crecimiento de miles de culturas independientes (robótica QFA, Tabla 2). Sin embargo, puesto QFA se basa en técnicas establecidas, de laboratorio tradicionales, sino que también puede llevarse a cabo mucho más barata mediante la sustitución de asistencia robot con pasos Manual (Manual de QFA, Tabla 2). Manual de QFA implica la replicación y la inoculación de los cultivos sobre discos de agar con una herramienta manual de pin y la transferencia manual de las placas hacia y desde una incubadora para la fotografía. El flujo de trabajo mismo análisis computacional puede ser aplicado para generar curvas de crecimiento de cualquier diseño experimental. QFA estimaciones salud física parecen ser relativamente precisa. En la Figura 4, las eficacias medias de cuatro grupos de ejemplo de supresiones de genes funcionalmente relacionados se destacan. La proximidad de los miembros de cada clase de genes relacionados funcionalmente en dos independenciarentes orígenes genéticos (ura3Δ y cdc13-1) indica la reproducibilidad de QFA estimaciones de fitness. Por ejemplo, sólo tres supresiones de genes (de un máximo de 4.300) separar a los tres miembros del complejo MRX conservada. De alto rendimiento alternativas a QFA para probar las características de crecimiento de cepas microbianas incluyen SGA 5,6, las pantallas de la competencia en las bibliotecas de código de barras y pantallas de captura de 11,12 cinética de la densidad óptica de lectores de placas espectrofotométrico 10, que se resumen en la Tabla 2 y se describe más detalladamente en otro lugar 8 . QFA y placas SGA, donde las culturas crecen en las superficies de agar agar (sólidos basados ​​en métodos, Cuadro 2) se puede manejar de forma rápida y fácil por el robot. Sólidos culturas superficie del agar son bien aireado durante todo el crecimiento y las células pueden crecer en cultivos fijos, permitiendo microbios sociables a crecer en una comunidad 14. Dejado a las comunidades microbianas intactas, unFECTOS sus propios micro-ambientes, toxinas que secretan tales como etanol, y posiblemente de señalización entre las células. Sin embargo, la continua mezcla de culturas líquidas, necesarias para lograr una aireación adecuada, de forma artificial altera las comunidades microbianas y sus entornos de microorganismos que puedan afectar a su modo de crecimiento. La contaminación cruzada es una preocupación menor en los métodos de agar sólido ya que hay menos oportunidad para que las salpicaduras de líquidos que transportan las células entre las culturas. Si se produce contaminación por el aire exterior transmitidas por microbios en los ensayos de agar sólido, a menudo puede ser detectada mediante inspección visual de las placas de agar sólido y representó o eliminado. Rendimiento QFA es significativamente mayor que la de paralelas métodos líquidos de dos maneras. En primer lugar, en QFA (y SGA) placas, cultivos inoculados se empaquetan con mayor densidad, lo que las culturas más independientes por placa. En QFA normalmente hay 308 culturas, sin contar las no experimentales culturas de borde (Figura 1) comcomparación en paralelo cultivos líquidos con 96 o 100 por placa culturas. En segundo lugar, los experimentos QFA se puede escalar a un número mucho mayor de las placas. Considerando que el número de placas analizadas en un único experimento QFA sólo está limitado por el espacio disponible en una sala de incubadora o caliente, la frecuencia mínima permisible de captura de imágenes y la frecuencia máxima alcanzable captura, el número de placas en un experimento de crecimiento líquido es fuertemente limitado por la capacidad del lector de placas (normalmente una o dos placas), o del apilador unido al lector de placas (típicamente 25-50 placas). Recientemente hemos llevado a cabo un experimento QFA totalmente automatizado en 123 platos, cada uno con 308 cultivos experimentales, dando 37.884 cultivos simultáneos. Se estima que el número máximo posible de las culturas de cultivo independientes en el líquido es de 96 (cultivos / placa) x 50 (placas / apilador) = 4,800, que es de alrededor de diez veces menor que nuestro rendimiento QFA. Una alternativa para las pantallas de cultivo líquidos es utilizar muchas gro automatizadowth dispositivos en paralelo (Tabla 1 de la revisión de Blomberg 8), cada uno con una capacidad de uno o dos platos. El control de temperatura en cada dispositivo es independiente, y así que las condiciones no son idénticas, pero suponiendo que lo sean, este flujo de trabajo que requieren al menos 190 dispositivos de este tipo para que coincida con el rendimiento QFA (suponiendo 200 cultivos líquidos por dispositivo). En resumen, QFA es un flujo de trabajo de alta calidad que pueden ser útiles para obtener los caracteres cuantitativos de crecimiento en los dos pequeños experimentos a escala centrados y pantallas de alto rendimiento. Es lo suficientemente flexible para ser aplicado eficazmente con diferentes requisitos para el equipo robótico. El componente de cálculo del flujo de trabajo QFA se basa en libre disposición, el código fuente abierto.

<table border="1"><tbody><tr><td> Nombre del reactivo / equipo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Replica Plater </td><td> Sigma </td><td> R-2508 </td><td> 96 pines manual de pintool con 1/8 pines de diámetro &quot; </td></tr><tr><td> Mezclar mate </td><td> Eppendorf </td><td> 5353 000.014 </td><td></td></tr><tr><td> Biomek FX </td><td> Beckman </td><td> A31842 </td><td></td></tr><tr><td> Teleshake </td><td> Thermo Scientific </td><td> 50095890 </td><td> Instalado en Biomek FX </td></tr><tr><td> BM3-SC </td><td> S &amp; P Robotics Inc </td><td> BM3-SC </td><td> 192 plataforma giratoria del carrusel </td></tr><tr><td> spImager </td><td> S &amp; P Robotics Inc </td><td> spImager </td><td> Manual de imagen de alta resolución </td></tr><tr><td> spImager con Cytomen </td><td> S &amp; P Robotics Inc </td><td> Costumbre </td><td> Temperatura controlada de imágenes automatizado de alta resolución </td></tr><tr><td> Cytomat 6001 con intercambiador de calor </td><td> Thermo Scientific </td><td> 51022222 </td><td> Se adjunta a spImager </td></tr><tr><td> Ecoline RE207 </td><td> Lauda </td><td> RE207 </td><td> Se adjunta a Cytomat </td></tr><tr><td> spImager con el carrusel de </td><td> S &amp; P Robotics Inc </td><td> Costumbre </td><td> Imagen automatizada de alta resolución </td></tr><tr><td> Pin Robot herramienta de accesorio para FP12pins </td><td> V &amp; P Científico </td><td> AFIX96FP12 </td><td> N / A </td></tr><tr><td> 96 x FP12 pines </td><td> V &amp; P Científico </td><td> FP12 </td><td> 50,4 mm de largo, 17 mm de longitud expuesta pin </td></tr><tr><td> Docking Station para la Herramienta de Pin </td><td> V &amp; P Científico </td><td> VP425 </td><td> Base de la estación de acoplamiento eliminado para permitir un secado de fans de los pasadores </td></tr><tr><td>Pin Cepillo de limpieza </td><td> V &amp; P Científico </td><td> VP425 </td><td> N / A </td></tr><tr><td> Replica Plater </td><td> Sigma </td><td> R-2508 </td><td> Herramienta manual pin </td></tr><tr><td> Nunc OmniTray con tapa </td><td> Nunc </td><td> 734-0490 </td><td> N / A </td></tr><tr><td> 96 así estériles placas de poliestireno </td><td> Greiner BioOne </td><td> 655161 </td><td> N / A </td></tr><tr><td> 96 así estériles tapas de poliestireno </td><td> Greiner BioOne </td><td> 656171 </td><td> N / A </td></tr></tbody></table> Tabla 3. Reactivos específicos y equipos.

a quantitative fitness analysis workflow

Análisis de aptitud cuantitativa (QFA) es un flujo de trabajo experimental y computacional para la comparación de eficacias de los cultivos microbianos crecido en paralelo 1,2,3,4. QFA se puede aplicar a las observaciones de las culturas centradas individuales, pero es más útil para todo el genoma interacción genética o la investigación de análisis de drogas a miles de culturas independientes. El método experimental central es la inoculación de independientes, diluidas cultivos líquidos microbianos sobre discos de agar sólidos que son incubados y se fotografiaron con regularidad. Las fotografías de cada punto de tiempo se analizan, produciendo cuantitativos estimaciones de la densidad de células, que se utilizan para construir curvas de crecimiento, lo que permite medidas cuantitativas aptitud para ser derivado. Eficacias Cultura se puede comparar a cuantificar y clasificar las fortalezas genéticas de interacción o de sensibilidad a los medicamentos. El efecto sobre la aptitud cultivo de cualquier tratamiento añadido en agar sustrato (por ejemplo, pequeñas moléculas, antibióticos o nutrientes) o se aplica a placas externoly (irradiación UV, por ejemplo, temperatura) se puede cuantificar por QFA. El flujo de trabajo QFA produce tasa de crecimiento estima análoga a los obtenidos por medición espectrofotométrica de paralelas cultivos líquidos en los lectores de placas de 96 pocillos o bien 200. Es importante destacar que QFA tiene el rendimiento significativamente mayor en comparación con estos métodos. Culturas QFA crecer en una superficie de agar sólido y por lo tanto bien aireado durante el crecimiento sin la necesidad de agitación o agitación. QFA rendimiento no es tan alta como la de algunos matriz sintética genética (SGA) de los métodos de detección 5,6. Sin embargo, ya que las culturas están fuertemente QFA diluido antes de ser inoculadas en agar, QFA puede capturar las curvas de crecimiento más completos, incluyendo las fases exponencial y la saturación de 3. Por ejemplo, las observaciones de la curva de crecimiento permiten tiempos de la cultura de duplicación que se calcula directamente con la alta precisión, como se indicó anteriormente 1. Aquí les presentamos unaprotocolo específico de QFA aplicado a miles de S. culturas cerevisiae, que se gestionan automáticamente mediante robots durante la inoculación, incubación y de imagen. Cualquiera de estos pasos automatizados puede ser sustituido por un procedimiento equivalente, manual, con una reducción asociada en el rendimiento, y que también presentan un protocolo de instrucciones rendimiento más bajo. Las mismas herramientas de software QFA se puede aplicar a las imágenes capturadas, ya sea en flujo de trabajo. Tenemos amplia experiencia en la aplicación de QFA a los cultivos de la levadura de gemación S. cerevisiae, pero esperamos que QFA será igualmente útil para examinar las culturas de la levadura de fisión S. pombe y cultivos bacterianos.

Watch this Scientific Journal Video about A Quantitative Fitness Analysis Workflow at JoVE.com

A Quantitative Fitness Analysis Workflow

Análisis de aptitud cuantitativa (QFA) es una serie complementaria de los métodos experimentales y computacionales para la estimación de eficacias de cultivos microbianos. QFA estima el efecto de mutaciones genéticas, medicamentos u otros tratamientos aplicados en el crecimiento de microbios. Los experimentos de escala a partir del análisis de las culturas individuales enfocada a miles de culturas paralelas se pueden diseñar.

Un flujo de trabajo de Análisis Cuantitativo Gimnasio

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Research

JoVE Journal

Biology

14.3K vistas.  Newcastle University Medical School. Análisis de aptitud cuantitativa (QFA) es una serie complementaria de los métodos experimentales y computacionales para la estimación de eficacias de cultivos microbianos. QFA estima el efecto de mutaciones genéticas, medicamentos u otros tratamientos aplicados en el crecimiento de microbios. Los experimentos de escala a partir del análisis de las culturas individuales enfocada a miles de culturas paralelas se pueden diseñar.

Video: A Quantitative Fitness Analysis Workflow

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A neuronales y astrocitos Co-cultivo de ensayo para el análisis de alto contenido de neurotoxicidad

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Investigación

Biología

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

Phosphoproteomics cuantitativo de ácidos grasos estimulada Saccharomyces cerevisiae</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Células vivas, imágenes y análisis cuantitativo de las células epiteliales embrionarias en Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Cuantitativa de células vivas por fluorescencia microscopia análisis de la levadura de fisión

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Análisis cuantitativo de la migración desordenada de las células mediante microscopía de Time-lapse vídeo

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Análisis cuantitativo de la autofagia mediante microscopía de fluorescencia avanzada 3D

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualización y Análisis de las moléculas de ARNm mediante fluorescencia<em&gt; In Situ</em&gt; La hibridación en<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Un ensayo de unión de alto rendimiento MHC II para el análisis cuantitativo del péptido epítopos

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Una nueva técnica de análisis cuantitativos de la pérdida del pelo en ratones Uso del análisis de escala de grises

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Flujo de trabajo completo para el análisis de las histonas modificaciones post-traduccionales El uso de espectrometría de masas de abajo hacia arriba: A partir de la histona Extracción de Análisis de Datos

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...