Wir danken allen Mitgliedern unseres Labors und dem Zentrum für Integrierte System-Biologie des Alterns und Ernährung (CISBAN) für die Unterstützung und hilfreiche Diskussionen. Diese Studie wurde vom Biotechnology and Biological Sciences Research Council (BBSRC) (BB/C008200/1) und des Wellcome Trust (075294, 093088) unterstützt.

<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>

Keine Interessenskonflikte erklärt.

QFA ist in vielerlei Hinsicht ein direkter Nachkomme von drei etablierten Labormaßstab mikrobiologischen Techniken: Kultur Verdünnungsreihe Stichproben 9, Wachstumskurve Bestimmung in belüfteten Flüssigkulturen 9 und 13 Replica-Plating. Diese drei Methoden werden zusammengefasst und verglichen mit QFA und andere High-Throughput-Techniken, die in Tabelle 2. Während Verdünnungsreihe Stichproben definieren, einem Stamm der Fitness als seine Fähigkeit, Kolonien in einer Reihe von Kulturen aus einer Reihe von Verdünnungen Inokulum gewachsen zu bilden, um QFA Maßnahmen Belastung Fitness durch wiederholte Beobachtungen einer einzigen Kultur konstruieren eine Wachstumskurve. Die Quantifizierung der Fitness von einzelnen Kulturen ermöglicht viele weitere Stämme gleichzeitig untersucht werden, unter identischen Bedingungen. Replizieren von Arrays der Kulturen ermöglicht wiederholtes Testen von Stammsammlungen, am sinnvollsten bei der Prüfung für Kultur Fitness Unterschiede in verschiedenen Umgebungen oder genetischen Hintergründen. Die QFA Protokoll vorgestellthier verwendet teure Roboter-Anlagen zu replizieren, beimpfen, inkubieren und Bild-Agar-Platten, geeignet für die Beobachtung Wachstum von Tausenden von unabhängigen Kulturen (Roboter QFA, Tabelle 2). Da jedoch QFA auf etablierten, traditionellen Labor-Techniken basiert, kann es auch viel billiger werden durch das Ersetzen Roboter mit Hilfe manueller Schritte (manuelle QFA, Tabelle 2) durchgeführt. Manuelle QFA beinhaltet Replikation und Inokulation der Kulturen auf Agarplatten mit einem manuellen Stiftwerkzeug und der manuellen Übertragung von Platten zu und von einem Inkubator für Fotografie. Das gleiche rechnerische Analyse Workflow angewendet werden, um Wachstumskurven von beiden experimentellen Design zu generieren. QFA Fitness Schätzungen zu sein scheinen vergleichsweise präzise. In Abbildung 4 sind die mittlere Fitness der vier Beispiel-Cluster von funktionell verwandten Gen-Deletionen hervorgehoben. Die räumliche Nähe der Mitglieder jeder funktionell verwandten Gen-Klasse in zwei unabhängigeHNO genetischen Hintergründen (ura3Δ und CDC13-1) zeigt die Reproduzierbarkeit der QFA Fitness-Schätzungen. Zum Beispiel, trennen nur 3 Gendeletionen (von möglichen 4300) die drei Mitglieder der konservierten MRX-Komplex. High-Throughput-Alternativen zu QFA zum Testen Wachstumseigenschaften mikrobielle Stämme umfassen SGA 5,6, Wettbewerb Bildschirme in Strichcode-Bibliotheken 11,12 und Bildschirme aufnehmenden optischen Dichte Kinetik spektrophotometrische Plattenleser 10, die in Tabelle 2 zusammengefasst sind, und an anderer Stelle ausführlicher beschrieben 8 . QFA und SGA-Platten, wo Kulturen wachsen auf Agar-Oberflächen (Agar-basierten Methoden, Tabelle 2) können schnell und einfach per Roboter behandelt werden. Solide Agaroberfläche Kulturen sind gut belüftet und das Wachstum im gesamten Zellen können in festen Kulturen wachsen, so gesellig Mikroben in einer Gemeinschaft zu 14 wachsen. Intakt gelassen, mikrobieller Gemeinschaften einUSWIRKUNG eigenen Mikroumgebung, sezernierenden Toxine, wie Ethanol, und möglicherweise Signalisierung zwischen Zellen. Allerdings kontinuierliches Mischen von flüssigen Kulturen, notwendig, um ausreichende Belüftung zu erreichen, künstlich stört mikrobieller Gemeinschaften und deren Mikro-Umgebungen, die ihre Funktionsweise Wachstum beeinträchtigen können. Cross-Kontamination ist weniger ein Problem in festen Agar-Methoden, da es weniger Gelegenheit für Flüssigkeitsspritzern tragenden Zellen zwischen den Kulturen. Wenn Kontamination durch fremde luftgetragenen Mikroben in festen Agar-Assays, kann es oft durch visuelle Inspektion von festen Agar-Platten erkannt werden und machten oder entfernt werden. QFA Durchsatz erheblich höher als die der Flüssigkeit parallel Verfahren auf zwei Arten. Zunächst auf QFA (und SGA)-Platten, inokuliert Kulturen zusammen dichter gepackt, so dass mehrere unabhängige Kulturen pro Platte. In QFA gibt es typischerweise 308 Kulturen, nicht mitgerechnet nicht-experimentellen Rand Kulturen (Abbildung 1) comVergleich zu flüssigen Kulturen mit 96 oder 100 Kulturen pro Platte parallel. Zweitens kann QFA Versuche zu einer viel größeren Anzahl von Platten skaliert werden. Während die Anzahl der Platten in einem einzigen Experiment analysiert QFA nur durch den verfügbaren Raum in einem Inkubator oder warmen Raum, der minimal zulässige Bilderfassungsvorrichtung Frequenz und der maximal erreichbaren Capture Frequenz beschränkt ist, ist die Anzahl von Platten in einem flüssigen Wachstumsmedium Experiment stark eingeschränkt durch die Kapazität der Platten-Lesegerät (typischerweise ein oder zwei Platten) oder der Ablage an der Platten-Lesegerät (typischer Wert von 25-50 Platten). Wir haben vor kurzem eine vollautomatische QFA Experiment auf 123 Platten mit je 308 experimentellen Kulturen durchgeführt, so dass 37.884 gleichzeitigen Kulturen. Wir schätzen, dass die maximal erreichbare Anzahl von unabhängigen Kulturen wachsen in flüssiger 96 (Kulturen / Platte) ist x 50 (Platten / Stapler) = 4800, die um das Zehnfache kleiner als unsere QFA Durchsatz ist. Eine Alternative für flüssige Kultur-Bildschirme ist für viele automatisierte gro nutzenwth Geräte parallel (Tabelle 1 aus der Bewertung von Blomberg 8), jede mit einer Kapazität von einem oder zwei Platten. Temperaturregelung in jedem Gerät ist unabhängig, und so Bedingungen sind nicht identisch, aber davon aus, dass sie sind, würde dies Workflow benötigen mindestens 190 solcher Geräte zu QFA Durchsatz (unter der Annahme 200 Flüssigkulturen pro Gerät) entsprechen. Zusammenfassend ist QFA eine hochwertige Workflow, sinnvoll angewendet werden kann, um quantitatives Wachstum Phänotypen in beiden Kleinen fokussiert Experimente und High-Throughput-Screens sammeln. Es ist flexibel genug, um effektiv mit unterschiedlichen Anforderungen an die Roboter-Anlagen angewendet werden. Die rechnerische Komponente der QFA Workflow basiert auf frei verfügbarer Open-Source-Code basiert.

<table border="1"><tbody><tr><td> Name des Reagenz / Ausrüstung </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Replica Plater </td><td> Sigma </td><td> R-2508 </td><td> 96 Pin manuelle pintool mit 1/8 &quot;Durchmesser Stifte </td></tr><tr><td> Mischen Sie 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> Installiert auf Biomek FX </td></tr><tr><td> BM3-SC </td><td> S &amp; P Robotics Inc </td><td> BM3-SC </td><td> 192 Regal drehenden Karussell </td></tr><tr><td> spImager </td><td> S &amp; P Robotics Inc </td><td> spImager </td><td> Hochauflösende Bildgebung manuelle </td></tr><tr><td> spImager mit Cytombei </td><td> S &amp; P Robotics Inc </td><td> Brauch </td><td> Temperaturgeführte automatisierte hochauflösende Bildgebung </td></tr><tr><td> Cytomat 6001 mit Wärmetauscher </td><td> Thermo Scientific </td><td> 51022222 </td><td> An spImager </td></tr><tr><td> Ecoline RE207 </td><td> Lauda </td><td> RE207 </td><td> An Cytomat </td></tr><tr><td> spImager mit Karussell </td><td> S &amp; P Robotics Inc </td><td> Brauch </td><td> Automatisierte hochauflösende Bildgebung </td></tr><tr><td> Roboter Pin Werkzeugaufnahme für FP12pins </td><td> V &amp; P Scientific </td><td> AFIX96FP12 </td><td> N / A </td></tr><tr><td> 96 x FP12 Stifte </td><td> V &amp; P Scientific </td><td> FP12 </td><td> 50,4 mm lang, 17 mm Stiftlänge ausgesetzt </td></tr><tr><td> Docking Station für Pin Tool </td><td> V &amp; P Scientific </td><td> VP425 </td><td> Docking-Station Basis entfernt, um Fan Trocknung von Nadeln kann </td></tr><tr><td>Pin-Reinigungsbürste </td><td> V &amp; P Scientific </td><td> VP425 </td><td> N / A </td></tr><tr><td> Replica Plater </td><td> Sigma </td><td> R-2508 </td><td> Manuelle Pin-Werkzeug </td></tr><tr><td> Nunc OmniTray mit Deckel </td><td> Nunc </td><td> 734-0490 </td><td> N / A </td></tr><tr><td> 96-Well-sterile Polystyrol-Platten </td><td> Greiner BioOne </td><td> 655161 </td><td> N / A </td></tr><tr><td> 96 Well Polystyrol sterilen Deckeln </td><td> Greiner BioOne </td><td> 656171 </td><td> N / A </td></tr></tbody></table> Tabelle 3. Reagenzien und Geräte.

a quantitative fitness analysis workflow

Quantitative Analyse Fitness (QFA) ist ein experimenteller und rechnerischer Workflow für den Vergleich von Fitness der mikrobiellen Kulturen parallel 1,2,3,4 gewachsen. QFA können fokussierten Beobachtungen der einzelnen Kulturen angewandt werden, sondern ist sehr nützlich für genomweite genetische Interaktion oder Drogen-Bildschirme der Untersuchung bis zu Tausenden von unabhängigen Kulturen. Die zentrale experimentelle Methode ist die Impfung von unabhängigen, verdünnte Flüssigkeit mikrobiellen Kulturen auf festen Agar-Platten, die bebrütet und werden regelmäßig fotografiert. Fotos von jedem Zeit-Punkt analysiert, Erstellung von quantitativen Zelldichte Schätzungen, die verwendet werden, um Wachstumskurven zu konstruieren, so dass quantitative Fitness-Maßnahmen abgeleitet werden. Kultur Fitnesswerte kann im Vergleich zu quantifizieren und zu zählen genetische Interaktion Stärken oder Drogen-Empfindlichkeiten werden. Die Auswirkungen auf die Kultur von den Behandlungen Fitness Hinzugefuegt in Substrat-Agar (z. B. kleine Moleküle, Antibiotika oder Nährstoffe) oder aufgebracht auf externen Plattenly (zB UV-Einstrahlung, Temperatur) kann durch QFA quantifiziert werden. Die QFA Workflow erzeugt Wachstum schätzt analog zu den durch spektrophotometrische Messung der parallelen flüssigen Kulturen in 96-Well oder 200-Well-Platte Software erhalten. Wichtig ist, daß QFA deutlich höheren Durchsatz im Vergleich mit solchen Methoden. QFA Kulturen wachsen auf einem festen Agar-Oberfläche und werden daher auch während des Wachstums ohne Rühren oder Schütteln belüftet. QFA Durchsatz ist nicht so hoch wie die einiger synthetischer Genetic Array (SGA) Screening-Verfahren 5,6. Da jedoch QFA Kulturen stark, bevor sie auf Agar geimpft verdünnt werden, können QFA erfassen vollständigere Wachstumskurven, einschließlich Exponential-und Sättigungs-Phasen 3. Zum Beispiel ermöglichen Wachstumskurve Beobachtungen Kultur Verdoppelungszeiten direkt mit hoher Genauigkeit geschätzt werden, wie zuvor diskutiert 1. Hier präsentieren wir einspezifische QFA-Protokoll angewandt, um Tausende von S. cerevisiae Kulturen, die automatisch durch Roboter sind während der Inokulation, Inkubation und Imaging behandelt. Jeder dieser Schritte kann automatisiert durch ein gleichwertiges, manuelle Verfahren ersetzt werden, mit einer zugehörigen Reduzierung der Durchlaufzeit, und wir präsentieren auch einen geringeren Durchsatz manuelle Protokoll. Die gleichen QFA Software-Tools, um Bilder in entweder Workflow erfasst angewendet werden. Wir haben umfangreiche Erfahrungen auf die Anwendung QFA Kulturen der Bäckerhefe S. cerevisiae, aber wir erwarten, dass QFA erweisen wird ebenso nützlich für die Prüfung Kulturen der Spalthefe S. pombe und Bakterienkulturen.

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

A Quantitative Fitness Analysis Workflow

Quantitative Analyse Fitness (QFA) ist eine ergänzende Reihe von experimentellen und rechnerischen Methoden zur Schätzung der mikrobiellen Kultur Fitnesswerte. QFA schätzt den Effekt genetischer Mutationen, Medikamente oder andere Behandlungen angewandt auf Mikrobenwachstum. Experimente Skalierung von fokussierten Analyse der einzelnen Kulturen zu Tausenden von parallelen Kulturen ausgelegt werden kann.

A Quantitative Analysis Fitness-Workflow

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 Aufrufe.  Newcastle University Medical School. Quantitative Analyse Fitness (QFA) ist eine ergänzende Reihe von experimentellen und rechnerischen Methoden zur Schätzung der mikrobiellen Kultur Fitnesswerte. QFA schätzt den Effekt genetischer Mutationen, Medikamente oder andere Behandlungen angewandt auf Mikrobenwachstum. Experimente Skalierung von fokussierten Analyse der einzelnen Kulturen zu Tausenden von parallelen Kulturen ausgelegt werden kann.

Serie: 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.

Eine Neuronale und Astrocyte Co-Kultur-Assay für High Content Analyse der Neurotoxizität

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

Forschung

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.

Quantitative Phosphoproteomics in Fatty Acid Stimulated 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.

Live-Cell-Imaging und Quantitative Analyse der embryonalen Epithelzellen in 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.

Quantitative Live Cell Fluoreszenz-Mikroskopie Analyse von 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 ...

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.

Quantitative Analyse der ungerichtete Migration von Zellen unter Verwendung Zeitraffer-Video-Mikroskopie

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.

Quantitative Analyse von Autophagie mit Advanced 3D Fluoreszenzmikroskopie

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.

Visualisierung und Analyse von mRNA Moleküle mittels Fluoreszenz<em&gt; In Situ</em&gt; Hybridisierung in<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.

Ein hoher Durchsatz MHC II-Bindungsassay für die quantitative Analyse von Peptid-Epitope

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.

Eine neue Technik zur quantitativen Analyse von Haarausfall in Mäuse mit Graustufen-Analyse

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.

Komplette Workflow zur Analyse von Histon-post-translationale Modifikationen Mit Bottom-up-Massenspektrometrie: Von Histone Extraktion in Datenanalyse

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