Name: a quantitative fitness analysis workflow
Uploaded: 2012-08-13T12:00:00
Duration: 11 min 39 s
Description: Watch this Scientific Journal Video about A Quantitative Fitness Analysis Workflow at JoVE.com

Agradecemos a todos os membros de nosso laboratório e do Centro de Biologia do Sistema Integrado de Envelhecimento e Nutrição (CISBAN) para apoio e discussões úteis. Este estudo foi apoiado pelo Biotechnology and Biological Sciences Research Council (BBSRC) (BB/C008200/1) e do 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>

Não há conflitos de interesse declarados.

QFA é em muitos sentidos, um descendente directo de três estabelecidas bancada em escala de técnicas microbiológicas: cultura de diluição testes da série 9 no local, a determinação da curva de crescimento em culturas líquidas gasosas 9 e revestimento réplica 13. Estes três métodos são resumidos e comparados com QFA e outras técnicas de alto rendimento na Tabela 2. Considerando que os ensaios de diluição no local da série definir aptidão uma estirpe como a sua capacidade para formar colónias em uma série de culturas crescidas a partir de uma gama de diluições de inóculo, QFA aptidão estirpe medidas por observações repetidas de uma única cultura para construir uma curva de crescimento. A quantificação aptidão a partir de culturas única permite muitas estirpes mais a ser examinados simultaneamente, sob condições idênticas. Replicando matrizes de culturas permite testes repetidos de coleções de tensão, mais útil ao testar as diferenças de cultura de fitness em ambientes diferentes ou fundos genéticos. O protocolo apresentado QFAaqui usa equipamento caro robótico para replicar, inocular, incubar e placas de agar de imagem, apropriado para a observação de crescimento de milhares de culturas independentes (QFA robótico, Tabela 2). No entanto, desde QFA baseia-se na estabelecidas, técnicas de laboratório tradicionais, pode também ser efectuada muito mais barato, substituindo assistência robô com passos manuais (Manual QFA, Tabela 2). Manual do QFA envolve replicação e inoculação de culturas em ágar usando uma ferramenta de pino manual e transferência manual de placas de e para uma incubadora para a fotografia. O fluxo de trabalho mesma análise computacional pode ser aplicado para gerar curvas de crescimento a partir de qualquer desenho experimental. Estimativas QFA aptidão parecem ser relativamente preciso. Na Figura 4, as aptidões médios de quatro exemplo, agregados de funcionalmente deleções de genes relacionados são realçados. A proximidade de membros de cada classe de genes funcionalmente relacionados em duas inderentes origens genéticas (ura3Δ e CDC13-1) indica a reprodutibilidade das estimativas QFA fitness. Por exemplo, apenas 3 a deleção do gene (de um 4.300 possível) separar os três membros do complexo MRX conservada. Alto rendimento alternativas para QFA para testar as características de crescimento de estirpes microbianas incluem PIG 5,6, telas de concorrência em bibliotecas de código de barras 11,12 e telas de captura cinética de densidade óptica em leitores espectrofotométricos placa 10, que estão resumidos na Tabela 2 e descrito mais completamente em outro lugar 8 . QFA e placas de PIG, onde as culturas crescem em superfícies sólidas de ágar (ágar métodos baseados, Tabela 2) pode ser tratada de forma rápida e fácil pelo robô. Culturas de superfície sólida de agar são bem arejadas todo o crescimento e as células podem crescer em culturas fixas, permitindo que os micróbios sociáveis ​​a crescer em uma comunidade 14. Deixado intacto, comunidades microbianas umffect suas próprias micro-ambientes, toxinas secretoras, tais como etanol, e possivelmente de sinalização entre as células. No entanto, contínua mistura de culturas líquidas, necessárias para alcançar aeração adequada, artificialmente perturba comunidades microbianas e os seus micro-ambientes que possam afetar seus modos de crescimento. A contaminação cruzada é uma preocupação menor em métodos de ágar sólido uma vez que há menos oportunidades para líquidos salpicados transportando células entre as culturas. Se ocorrer contaminação por estrangeiros transportados pelo ar micróbios em ensaios de agar sólidos, que muitas vezes podem ser detectado por inspecção visual das placas de agar sólido e representaram ou removido. Rendimento QFA é significativamente mais elevada do que a de paralelas métodos líquidos de duas maneiras. Primeiro de tudo, no QFA (e SGA) placas, culturas inoculadas são embalados em conjunto de forma mais densa, dando culturas mais independentes por placa. Em QFA existem normalmente 308 culturas, não contando com culturas não experimentais de ponta (Figura 1) COMcomparação em paralelo culturas líquidas com 96 ou 100 culturas por placa. Em segundo lugar, as experiências QFA pode ser dimensionada para um número muito maior de pratos. Considerando que o número de placas analisados ​​numa experiência QFA único é limitada apenas pelo espaço disponível na sala de incubadora ou quente, a frequência de captura de imagem e mínima permitida a frequência de captura máxima alcançável, o número de placas em uma experiência de crescimento líquido é fortemente limitada pela capacidade do leitor de placas (tipicamente um ou dois pratos), ou do empilhador ligado ao leitor de placas (tipicamente 25-50 placas). Recentemente, realizou uma experiência totalmente automatizado QFA em 123 placas, cada uma com 308 culturas experimentais, dando 37,884 culturas simultâneas. Nós estimamos que o número máximo possível de culturas independentes que crescem em líquido é 96 (culturas / placa) x 50 (placas / empilhador) = 4800, que é de cerca de dez vezes menor do que o nosso rendimento QFA. Uma alternativa para as telas de cultura líquido é utilizar muitos automatizado grodispositivos wth em paralelo (Tabela 1 a partir da revisão por Blomberg 8), cada um tendo uma capacidade de um ou dois pratos. Controle de temperatura em cada dispositivo é independente, e assim as condições não são idênticas, mas assumindo que eles são, este fluxo de trabalho exigiria pelo menos 190 desses dispositivos para combinar QFA rendimento (assumindo 200 culturas líquidas por dispositivo). Em resumo, QFA é um fluxo de trabalho de alta qualidade que podem ser utilmente aplicadas para reunir fenótipos quantitativos de crescimento em ambos os experimentos de pequena escala e centraram-se alto rendimento telas. É flexível o suficiente para ser aplicado de forma eficaz com diferentes requisitos para o equipamento robótico. O componente computacional do fluxo de trabalho QFA baseia-se livremente disponível código fonte, aberto.

<table border="1"><tbody><tr><td> Nome de reagente / equipamentos </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Replica Plater </td><td> Sigma </td><td> R-2508 </td><td> 96 pinos Manual pintool com 1/8 &quot;de diâmetro pinos </td></tr><tr><td> Misture-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 em Biomek FX </td></tr><tr><td> BM3-SC </td><td> S &amp; P Robotics Inc </td><td> BM3-SC </td><td> 192 prateleira rotativa carrossel </td></tr><tr><td> spImager </td><td> S &amp; P Robotics Inc </td><td> spImager </td><td> Alta resolução manual de imagem </td></tr><tr><td> spImager com Cytomem </td><td> S &amp; P Robotics Inc </td><td> Personalizado </td><td> Temperatura controlada alta resolução de imagem automatizado </td></tr><tr><td> Cytomat 6001 com trocador de calor </td><td> Thermo Scientific </td><td> 51022222 </td><td> Anexado ao spImager </td></tr><tr><td> Ecoline RE207 </td><td> Lauda </td><td> RE207 </td><td> Anexado ao Cytomat </td></tr><tr><td> spImager com carrossel </td><td> S &amp; P Robotics Inc </td><td> Personalizado </td><td> Resolução de imagem automatizado de alta </td></tr><tr><td> Robot fixação ferramenta pinos para FP12pins </td><td> V &amp; P Científico </td><td> AFIX96FP12 </td><td> N / A </td></tr><tr><td> 96 x FP12 pinos </td><td> V &amp; P Científico </td><td> FP12 </td><td> 50,4 mm de comprimento, 17 mm de comprimento exposto pin </td></tr><tr><td> Docking Station para a Ferramenta de Pin </td><td> V &amp; P Científico </td><td> VP425 </td><td> Encaixe de estação de base removido para permitir a secagem do ventilador de pinos </td></tr><tr><td>Pin Escova de limpeza </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> Ferramenta manual de pinos </td></tr><tr><td> Nunc OmniTray com Tampa </td><td> Nunc </td><td> 734-0490 </td><td> N / A </td></tr><tr><td> 96 bem estéreis placas de poliestireno </td><td> Greiner BioOne </td><td> 655161 </td><td> N / A </td></tr><tr><td> 96 bem estéreis tampas de poliestireno </td><td> Greiner BioOne </td><td> 656171 </td><td> N / A </td></tr></tbody></table> Tabela 3. Reagentes específicos e equipamentos.

a quantitative fitness analysis workflow

Análise Quantitativa de Fitness (QFA) é um fluxo de trabalho experimental e computacional para comparar as aptidões de culturas microbianas cultivadas em 1,2,3,4 paralelo. QFA pode ser aplicado a observações orientadas de culturas individuais, mas é mais útil para genoma interacção genética ou telas de drogas investigar até milhares de culturas independentes. O método central experimental é a inoculação de independentes, diluir líquidos culturas microbianas em placas de agar sólidos que são incubadas e regularmente fotografadas. Fotografias de cada ponto do tempo são analisados, produzindo estimativas quantitativas de células de densidade, que são utilizados para construir curvas de crescimento, permitindo que as medidas quantitativas aptidão para ser derivado. Aptidões de cultura podem ser comparados para quantificar e classificar forças de interacção genética ou sensibilidades de drogas. O efeito sobre a aptidão da cultura de quaisquer tratamentos adicionados em agar de substrato (por exemplo, moléculas pequenas, antibióticos ou nutrientes) ou aplicadas a placas de externoly (por exemplo, irradiação UV, temperatura) pode ser quantificado por QFA. O fluxo de trabalho QFA produz taxa de crescimento estima análoga aos obtidos por medição espectrofotométrica da paralelas culturas líquidas em leitores de placas de 96 poços ou de 200 poços. Importante, QFA tem rendimento significativamente superior em comparação com esses métodos. Culturas QFA crescer sobre uma superfície de agar sólido e são, portanto, bem arejadas durante o crescimento sem a necessidade de agitação ou agitação. Rendimento QFA não é tão alto quanto o de alguns matriz genético sintético (SGA) métodos de triagem 5,6. No entanto, uma vez que as culturas QFA são fortemente diluído antes de ser inoculadas em ágar, QFA pode capturar as curvas de crescimento mais completos, incluindo exponencial e fases de saturação 3. Por exemplo, as observações da curva de crescimento permitir tempos de duplicação de cultura a ser estimado directamente com alta precisão, como discutido anteriormente 1. Aqui apresentamos umaespecífica QFA protocolo aplicado a milhares de S. cerevisiae culturas que são automaticamente tratados por robôs durante a incubação a inoculação, e de imagem. Qualquer um destes passos automatizados pode ser substituído por um procedimento, o equivalente manual, com uma redução na taxa de transferência associada, e também apresenta um protocolo manual inferior de transferência. As mesmas ferramentas de software QFA pode ser aplicado a imagens capturadas em qualquer fluxo de trabalho. Temos uma vasta experiência QFA aplicando às culturas da levedura de brotamento S. cerevisiae, mas esperamos que QFA provará igualmente útil para examinar as culturas da levedura S. pombe e as culturas bacterianas.

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

A Quantitative Fitness Analysis Workflow

Análise Quantitativa de Fitness (QFA) é uma série complementar de métodos experimentais e computacionais para estimar as aptidões cultura microbiana. QFA estima o efeito de mutações genéticas, drogas ou outros tratamentos aplicados sobre o crescimento micróbio. Experimentos de escala a partir da análise focalizada de uma única cultura a milhares de culturas paralelas podem ser projetados.

Um fluxo de trabalho Análise Quantitativa de Fitness

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

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 Neuronal e astrocitária Ensaio Co-Cultura de Análise de Conteúdo de Alta de neurotoxicidade

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

Biologia

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 quantitativa em ácidos graxos 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.

Em células vivas, Imagem e Análise Quantitativa de células embrionárias epiteliais em 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.

Quantitativas de células vivas de Análise de microscopia de fluorescência de levedura

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.

Análise Quantitativa de Migração aleatória de células usando microscopia de lapso de tempo 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.

Análise Quantitativa de Autophagy usando microscopia de fluorescência avançada 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, ...

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.

Um Ensaio de Ligação de Alto Rendimento II do MHC para análise quantitativa de Peptide Epitopes

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.

Uma nova técnica de Análise Quantitativa de perda de cabelo em ratos usando Análise Tons de Cinza

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.

Fluxo de trabalho completo para Análise de histona modificações pós-traducionais Usando Bottom-up Espectrometria de Massa: De histona Extração de Análise de Dados

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

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.