Nous tenons à remercier tous les membres de notre laboratoire et le Centre pour la biologie Système intégré de vieillissement et de la nutrition (CISBAN) pour le soutien et les discussions utiles. Cette étude a été soutenue par la biotechnologie et sciences biologiques du Conseil de recherche (BBSRC) (BB/C008200/1) et le 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>

Pas de conflits d&#39;intérêt déclarés.

QFA est à bien des égards un lien direct descendant de trois établis l&#39;échelle du laboratoire des techniques microbiologiques: la culture des tests de dilution de la série 9, place de détermination courbe de croissance dans des cultures liquides aérés 9 et placage réplique 13. Ces trois méthodes sont résumés et comparés avec QFA et d&#39;autres techniques à haut débit dans le tableau 2. Alors que les tests de dilution de la série comptant définir de remise en forme d&#39;une souche que sa capacité à former des colonies dans une série de cultures cultivées à partir d&#39;une gamme de dilutions d&#39;inoculum, de remise en forme QFA souche mesures par des observations répétées d&#39;une culture unique pour construire une courbe de croissance. Quantification de remise en forme à partir de cultures en un seul permet de nombreuses souches plus à être examinés simultanément, dans des conditions identiques. Réplication des tableaux de cultures permet des tests répétés de collections de souches, la plupart utilement lors de l&#39;essai pour les différences de conditionnement physique de la culture dans différents environnements ou antécédents génétiques. Le protocole présenté QFAutilise ici coûteux équipement robotique pour reproduire, ensemencer, incuber et des plaques d&#39;agar d&#39;image, approprié pour observer la croissance des milliers de cultures indépendantes (robotique QFA, tableau 2). Cependant, depuis QFA est basé sur des techniques de laboratoire établies, traditionnels, il peut également être réalisée beaucoup plus cher en remplaçant l&#39;aide de robot avec des étapes manuelles (manuel QFA, tableau 2). Manuel QFA implique la réplication et l&#39;inoculation des cultures sur des plaques de gélose en utilisant un outil de broche manuel et le transfert manuel des plaques vers et à partir d&#39;un incubateur pour la photographie. Le flux de travail même analyse computationnelle peut être appliquée pour générer des courbes de croissance de soit la conception expérimentale. QFA estimations de conditionnement physique semblent être relativement précis. Dans la figure 4, les fitnesses moyennes de quatre grappes par exemple de la délétion de gènes fonctionnellement liés sont mis en évidence. La proximité des membres de chaque classe fonctionnellement lié au gène de dans deux indépendancehorizons différents (génétiques ura3Δ et CDC13-1) indique la reproductibilité des estimations de remise en forme QFA. Par exemple, seulement 3 délétions de gènes (sur un 4300 possible) séparer les trois membres du complexe MRX conservée. À haut débit alternatives à QFA pour l&#39;essai des caractéristiques de croissance des souches microbiennes comprennent SGA 5,6, écrans de la concurrence dans les bibliothèques à code-barres et des écrans de saisie 11,12 cinétique de densité optique dans les lecteurs de plaques spectrophotométriques 10, qui sont résumées dans le tableau 2 et décrits plus en détail ailleurs 8 . QFA et les plaques SGA, où les cultures se développent sur ​​des surfaces de gélose solide (gélose des méthodes basées sur, tableau 2) peuvent être traitées rapidement et facilement par un robot. Solides cultures de surface de gélose sont bien aérées long de la croissance et les cellules peuvent croître dans les cultures fixes, permettant microbes sociables à grandir dans une communauté 14. Laissée intacte, les communautés microbiennes quelquesffect leurs propres micro-environnements, sécrétant des toxines comme l&#39;éthanol, et éventuellement de signalisation entre les cellules. Cependant, continue le mélange des cultures liquides, nécessaires pour atteindre une aération adéquate, artificiellement perturbe les communautés microbiennes et leurs micro-environnements qui peuvent influer sur leurs modes de croissance. La contamination croisée est moins un sujet de préoccupation dans les méthodes de gélose solide car il ya moins de possibilités de projections de liquides transportant des cellules entre les cultures. Si la contamination se produit par l&#39;étranger suspension dans l&#39;air des microbes dans les dosages de gélose solide, il peut souvent être détectés par une inspection visuelle des plaques d&#39;agar solides et ont représenté ou supprimés. QFA débit est sensiblement plus élevée que celle de méthodes parallèles liquides de deux manières. Tout d&#39;abord, sur la QFA (et SGA) plaques, cultures inoculées sont emballés ensemble plus dense, donnant des cultures plus indépendants par plaque. En QFA il ya généralement 308 cultures, sans compter les cultures de bord non-expérimentales (Figure 1) comcomparativement à des cultures liquides parallèle avec 96 ou 100 cultures par plaque. Deuxièmement, les expériences QFA peut être adapté à un plus grand nombre de plaques. Alors que le nombre de plaques analysés en une seule expérience QFA est seulement limité par l&#39;espace disponible dans une salle incubateur ou chaud, la fréquence minimale admissible de capture d&#39;image et de la fréquence de capture maximale possible, le nombre de plaques dans une expérience de croissance liquide est fortement limitée par la capacité du lecteur de plaque (typiquement une ou deux plaques), ou de l&#39;empileur fixé à la plaque de lecteur (typiquement 25-50 plaques). Nous avons récemment réalisé une expérience entièrement automatisé QFA sur 123 plaques, chacune avec 308 cultures expérimentales, ce qui donne 37,884 cultures simultanées. Nous estimons que le nombre maximum possible des cultures indépendantes qui poussent dans liquide est 96 (cultures / plaque) x 50 (plaques / empileur) = 4800, ce qui représente environ dix fois moins que notre débit QFA. Une alternative pour les écrans de culture liquide est d&#39;utiliser un grand nombre gro automatiséwth dispositifs en parallèle (Tableau 1 de la revue par Blomberg 8), chacun ayant une capacité de un ou deux plaques. Contrôle de la température dans chaque dispositif est indépendant, et ainsi de conditions ne sont pas identiques, mais en supposant qu&#39;ils sont, ce flux de travail, il faudrait au moins 190 de ces dispositifs pour correspondre à un débit QFA (en supposant que 200 cultures liquides par appareil). En résumé, QFA est un workflow de haute qualité qui peuvent être utilement appliquées à recueillir phénotypes de croissance quantitative dans les deux petites expériences à l&#39;échelle axés et à haut débit écrans. Il est suffisamment souple pour être appliqué efficacement avec différentes exigences pour l&#39;équipement robotique. La composante de calcul du flux de travail QFA est basé sur librement disponible, le code source ouvert.

<table border="1"><tbody><tr><td> Nom de réactif / équipements </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Réplique Plater </td><td> Sigma </td><td> R-2508 </td><td> 96 broches manuel pintool avec 1/8 po de diamètre broches </td></tr><tr><td> Mélanger Maté </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> Installé sur Biomek FX </td></tr><tr><td> BM3-SC </td><td> S &amp; P Robotics Inc </td><td> BM3-SC </td><td> 192 plateau rotatif carrousel </td></tr><tr><td> spImager </td><td> S &amp; P Robotics Inc </td><td> spImager </td><td> Haute résolution manuelle d&#39;imagerie </td></tr><tr><td> spImager avec Cytomà </td><td> S &amp; P Robotics Inc </td><td> Coutume </td><td> Température contrôlée automatisé imagerie à haute résolution </td></tr><tr><td> Cytomat 6001 avec échangeur de chaleur </td><td> Thermo Scientific </td><td> 51022222 </td><td> Rattaché à spImager </td></tr><tr><td> Ecoline RE207 </td><td> Lauda </td><td> RE207 </td><td> Rattaché à Cytomat </td></tr><tr><td> spImager avec carrousel </td><td> S &amp; P Robotics Inc </td><td> Coutume </td><td> Automatisé imagerie à haute résolution </td></tr><tr><td> Luminaire Robot outil de broche pour FP12pins </td><td> V &amp; P scientifique </td><td> AFIX96FP12 </td><td> N / A </td></tr><tr><td> 96 x FP12 broches </td><td> V &amp; P scientifique </td><td> FP12 </td><td> 50,4 mm de long, 17 mm longueur de tige exposée </td></tr><tr><td> Station d&#39;accueil pour l&#39;outil de Pin </td><td> V &amp; P scientifique </td><td> VP425 </td><td> Station de base d&#39;accueil retiré pour permettre le séchage de fans de broches </td></tr><tr><td>Pin Brosse de nettoyage </td><td> V &amp; P scientifique </td><td> VP425 </td><td> N / A </td></tr><tr><td> Réplique Plater </td><td> Sigma </td><td> R-2508 </td><td> Outil de broche Manuel </td></tr><tr><td> Nunc OmniTray avec couvercle </td><td> Nunc </td><td> 734-0490 </td><td> N / A </td></tr><tr><td> 96 plaques de polystyrène et stériles </td><td> Greiner BioOne </td><td> 655161 </td><td> N / A </td></tr><tr><td> 96 couvercles de polystyrène et stériles </td><td> Greiner BioOne </td><td> 656171 </td><td> N / A </td></tr></tbody></table> Tableau 3. Réactifs et équipements spécifiques.

a quantitative fitness analysis workflow

Analyse quantitative de remise en forme (QFA) est un flux de travail expérimental et de calcul pour comparer fitnesses de cultures microbiennes cultivées en 1,2,3,4 parallèle. QFA peut être appliquée à des observations ciblées de cultures en un seul, mais est très utile pour l&#39;ensemble du génome d&#39;interaction génétique ou de dépistage de drogues de l&#39;enquête jusqu&#39;à des milliers de cultures indépendantes. La méthode expérimentale centrale est l&#39;inoculation des indépendants, diluer le liquide cultures microbiennes sur des plaques d&#39;agar solides qui sont incubés et régulièrement photographié. Photographies de chaque point dans le temps sont analysés, la production des estimations quantitatives de densité de cellules, qui sont utilisés pour construire des courbes de croissance, ce qui permet des mesures de remise en forme quantitatifs pour être dérivée. Fitnesses Culture peut être comparé à quantifier et de classer les forces d&#39;interaction génétiques ou sensibilités aux médicaments. L&#39;effet sur l&#39;aptitude de la culture de tous les traitements ajouté dans la gélose substrat (par exemple les petites molécules, des antibiotiques ou des nutriments) ou appliqués sur des plaques externesment (par exemple l&#39;irradiation UV, température) peut être quantifiée par QFA. Le flux de travail QFA produit le taux de croissance les estimations analogues à ceux obtenus par mesure spectrophotométrique de parallèles cultures liquides dans les lecteurs de plaques à 96 puits ou 200 puits. Il est important, QFA a un débit nettement plus élevé par rapport à ces méthodes. Cultures QFA croître sur une surface de la gélose solide et sont donc bien aéré pendant la croissance sans la nécessité d&#39;agitation ou secousses. Débit QFA n&#39;est pas aussi élevé que celui de certains antenne synthétique génétique (SGA) les méthodes de dépistage 5,6. Cependant, puisque les cultures QFA sont fortement dilués avant d&#39;être ensemencé sur une gélose, QFA peut capturer des courbes de croissance plus complètes, y compris les phases de saturation exponentielle et 3. Par exemple, les observations des courbes de croissance permettent des temps de doublement de la culture à estimer directement avec une grande précision, tel que discuté précédemment 1. Nous présentons ici unespécifique QFA protocole appliqué à des milliers de S. cultures cerevisiae qui sont automatiquement gérés par des robots lors de l&#39;inoculation, l&#39;incubation et l&#39;imagerie. N&#39;importe laquelle de ces étapes automatisées peuvent être remplacés par un équivalent, une procédure manuelle, avec une réduction du débit associé, et nous présentons également un protocole inférieure débit manuel. Les mêmes outils logiciels QFA peut être appliqué aux images capturées dans les deux flux de travail. Nous avons une vaste expérience application QFA à des cultures de la levure bourgeonnante S. cerevisiae, mais nous nous attendons à ce que QFA va se révéler tout aussi utile pour examiner les cultures de la levure à fission S. pombe et des cultures bactériennes.

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

A Quantitative Fitness Analysis Workflow

Analyse quantitative de remise en forme (QFA) est une série complémentaire de méthodes expérimentales et de calcul pour estimer fitnesses cultures microbiennes. QFA estime l&#39;effet de mutations génétiques, les médicaments ou autres traitements appliqués sur la croissance des microbes. Mise à l&#39;échelle des expériences de l&#39;analyse ciblée des cultures en un seul à des milliers de cultures parallèles peuvent être conçus.

Un flux de travail de remise en forme Analyse quantitative

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 vues.  Newcastle University Medical School. Analyse quantitative de remise en forme (QFA) est une série complémentaire de méthodes expérimentales et de calcul pour estimer fitnesses cultures microbiennes. QFA estime l'effet de mutations génétiques, les médicaments ou autres traitements appliqués sur la croissance des microbes. Mise à l'échelle des expériences de l'analyse ciblée des cultures en un seul à des milliers de cultures parallèles peuvent être conçus.

Vidéo: 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.

Un neuronale et astrocytes de co-culture de dosage pour l&#39;analyse du contenu haute de la neurotoxicité

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

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Biologie

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.

Phosphoprotéomique quantitatives en acide gras Stimulé 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.

Imagerie des cellules vivantes et de l&#39;analyse quantitative des cellules embryonnaires épithéliales 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.

Quantitatives de fluorescence Live Cell-microscopie Analyse de la levure à fission

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.

Analyse quantitative des migrations au hasard des cellules en utilisant la microscopie vidéo time-lapse

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.

Analyse quantitative de l&#39;autophagie en utilisant avancée Fluorescence Microscopy 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.

Visualisation et d&#39;analyse de molécules d&#39;ARNm par fluorescence<em&gt; In Situ</em&gt; Hybridation dans<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 essai de liaison à haut débit CMH II pour l&#39;analyse quantitative du peptide épitopes

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.

Une nouvelle technique pour l&#39;analyse quantitative de la perte de cheveux chez la souris Utilisation de l&#39;analyse des niveaux de gris

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.

Flux de travail complet pour l&#39;analyse des histones modifications post-traductionnelles utilisant la spectrométrie de masse Bottom-up: De Histone Extraction à l&#39;analyse des données

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