Agradecemos ao Centro de Desenvolvimento de Imagens Biológicas (BIDC) da Universidade da Califórnia, San Francisco para o uso do Imaris, XT Imaris e Matlab. Agradecemos a V. Kharazia para a assistência técnica e para Henry, LK Floren, L. Daitch por suas contribuições para a edição do manuscrito. Este trabalho foi financiado por fundos do Estado da Califórnia Medical Research em Álcool e Abuso de Substâncias através UCSF a SEB, dos Institutos Nacionais de Saúde: 1R21DA029966-01 e NIH prêmio Fast Track para a tela a coleção MLSMR a SEB, UCSF Faculdade de Farmácia ( Escritório de Dean e Farmácia Clínica) e da Faculdade de Medicina (Farmacologia Clínica e Terapêutica Experimental) para CLHK.

1. Tridimensional Análise morfométrica de uma única célula alterações fenotípicas <ol><li> De rim embrionário humano (HEK293) as células foram transfectadas com a hemaglutinina (HA), factor de libertação da corticotropina marcado receptor-2 (CRF-R2), um receptor acoplado à proteína G (GPCR), como descrito anteriormente 4, 5. </li><li> As células foram deixadas sem tratamento (sem tratamento, NT), estimuladas com o ligando endógeno do CRF-R2, factor de libertação de corticotropina, CRF (1 uM, 30 min), ou pré-tratados com um antagonista de CRF-R2 selectiva, anti-sauvagina 30 (AS -30, 1 uM, 30 min) antes do tratamento com o agonista. </li><li> As células foram então fixadas, permeabilizadas e tratados com anti-HA. CRF-R2 foi visualizada utilizando Alexa 594 nm conjugado anti-ratinho (IgG 1) anticorpo. DAPI foi utilizado para visualizar a fase mitótica núcleos. </li><li> Para limitar a subjectividade experimentador, as condições experimentais não foram conhecidos depois as imagens foram obtidas e analisadas. </li><li> Adquirimos imagems fixos a partir de células HEK293, utilizando um plano-Apochromat 63x/1.4 óleo objectivo DIC e Zeiss LSM 510 META microscópio confocal ligado a um sistema de laser Coherent integrado de dois fotões compreende um laser Verdi-V5 e um 900-F Mira sistema laser. </li><li> Durante o processo de aquisição de dados, as células foram tanto compartimentado por seccionamento multiespectral, 488 nm, e 790 nm (350 nm ~ Ex 2PH.) E particionamento de z-(em incrementos de 0,5 ^ M) de modo a incluir os dados a partir da membrana nuclear para o receptor externo extracelular extremidades. </li><li> Os dados de fluorescência foram primeiro tratados com Imaris, que permite a visualização e segmentação do conjunto de dados de microscopia 3D, e um modelo 3D composto por voxels cúbicos foi criado para análise morfométrica. Então, Imaris XT módulo foi utilizado para Imaris interface com MATLAB linguagem de programa de computador para determinar o ponto de coordenadas das extensões de GPCR. Para levar em conta a variabilidade celular, que obteve e analisou as imagens de fluorescência taken das 22 células: sem tratamento (NT) (n = 7), o tratamento com agonista (CRF) (n = 8) e um pré-tratamento com o antagonista (AS-30) antes de tratamento com o agonista (n = 7) (Figura 2) . <ol><li> A região de interesse (ROI) deve incluir uma célula que não está em fase mitótica ativa e não perto de outras células. Deste modo, a análise inclui as células com apenas um núcleo e as extensões do receptor não são perturbados pela proximidade de outras células. </li><li> A estrutura 3D celular foi reconstruída a partir de dados de fluorescência utilizando multiespectrais Imaris (v.7.1.1). </li><li> Seguindo o algoritmo desenvolvido por Imaris, primeiro processamento da superfície foi utilizada para representar a membrana nuclear. Imaris irá determinar se existe mais do que um núcleo na ROI. </li><li> Em seguida, o algoritmo de criação de pontos foi utilizada para localizar a extensão CRF-R2. Detecção manchas foi utilizada porque compensa o ruído de fundo e da intensidade da irregularrede complexa de células em forma amorfa. </li><li> Para maximizar a inclusão de cada unidade de detecção de fluorescência de CRF-R2, o diâmetro dos locais foi fixado a 0,2 um, o que é a unidade mais pequena dentro da imagem para extrapolar informações distintas sob a forma de uma intensidade medida utilizando um filtro de Gauss. Filtragem local foi incorporado no processo de criação de pontos automatizado. O software, no entanto, dá ao usuário a flexibilidade de usar filtros para definir os parâmetros. </li><li> Para evitar o truncamento de dados, o conjunto de dados foi convertido a partir de 8 bits (unsigned) ponto fixo, a 32-bit decimal. </li><li> Os dados voxel intensidades foram trocadas para detectar dados de coordenadas usando Imaris módulo XT interface com MATLAB e a localização exacta de cada ponto espacial foi determinada através da realização de uma transformação distância utilizando a membrana nuclear, tal como um ponto de referência (Figura 3). </li></ol></li><li> Os dados resultantes podem ser quantificados e apresentados em formato gráfico para sanálise tatistical. As comparações entre os grupos foram realizadas utilizando ANOVA two-way e Bonferroni pós-teste. Os dados são apresentados como média ± DP. As diferenças são consideradas significativas quando p * &lt;0,05. Os cálculos foram feitos com o programa GraphPad Prism 5.02 (Figura 4). </li></ol> 2. Resultados representativos Para demonstrar o poder da nossa abordagem, quantificou as alterações celulares que resultam da interacção da proteína G receptores acoplados (GPCRs) e factor de libertação da corticotropina receptor-2 (CRF-R2) com o seu ligando endógeno de CRF em células HEK293 transfectadas. Mostra-se que o CRF-R2 Os receptores estão localizados na membrana plasmática e do projecto de regiões finitas da membrana das células (Figura 2A e Filme 1). Usando a análise 2D convencional, é possível detectar este subconjunto de extracelulares receptores CRF-R2 apenas se analisar o receptor de adesão points no vidro cobre. Consequentemente perdemos qualquer outra informação derivada de z-empilhados dados multiespectrais (Figura 5). Quando as células são tratadas com CRF, os receptores extracelulares são muito reduzidos, tal como demonstrado pela diminuição da distância dos pontos da membrana plasmática. Eles também são redistribuídas principalmente a partir de locais em um número finito de locais discretos (Figura 2B e Filme 2). O efeito do CRF sobre a distribuição dos receptores da membrana é evitada por pré-tratamento com o antagonista de CRF-R2 específico, antisavagine 30 (AS-30) e descobrimos que as extensões de CRF-R2 não mudam (Figura 2C e Filme 3). A distribuição de pontos distal, representada nas 5 um espectro de cores codificadas com intervalos, é utilizada para visualizar a distância dos voxels da membrana nuclear. Não pretreatm tratamento e antagonistaent (AS-30, 1 ^ M, 30 min) antes de tratamento com o agonista (CRF, 1 uM, 30 min) não mostram diferenças (ns) significativa na contracção GPCR. O tratamento das células com o agonista (CRF, 1 uM, 30 min) reduz progressivamente o número de CRF-R2 contendo voxels quando comparado com qualquer outro tratamento, 0-5 ^ m (ns), 6-15 uM (** p &lt; 0,01) e&gt; 15 ^ m (*** p &lt;0,005) ou em comparação com o tratamento AS-30, 0-10 ^ m (ns), 11-15 ^ m (** p &lt;0,01) e&gt; 15 ^ m (*** p &lt;0,005) (Figura 4). <img alt="Figura 1" src="/files/ftp_upload/4233/4233fig1.jpg" /> Figura 1. Diagrama das técnicas atuais disponíveis e sua limitação para analisar imagens de fluorescência. <a href="http://www.jove.com/files/ftp_upload/4233/4233fig1large.jpg" target="_blank">Clique aqui para ver maior figura</a> . <img alt="Figura 2" src="/files/ftp_upload/4233/4233fig2.jpg" /> <strong&gt; Figura 2. 3D multiespectrais imagens de fluorescência das células HEK293 transfectadas com HA-CRF-R2, antes e depois do tratamento agonista e antagonista. Imagens fundidas mostrando HA-CRF-R2-sondadas com anti-HA e visualizados utilizando Alexa 488 de anticorpo anti-ratinho (IgG 1) conjugado secundário; DAPI foi utilizado para visualizar os núcleos. As imagens foram obtidas com microscópio confocal de varredura a laser (CLS). Escala da barra 5 um. <img alt="Figura 3" src="/files/ftp_upload/4233/4233fig3.jpg" /> Figura 3. Modelo 3D do HEK293 células transfectadas com HA-CRF-R2 reconstruído a partir de imagens usando o software CLS Imaris. Rendering superfície do núcleo e da criação de pontos que descreve extensões GPCR convertidos em pequenas vesículas. Os dados de fluorescência foram primeiro tratados com Imaris que permite a visualização e segmentação do conjunto de dados 3D de microscopia. Então, Imaris XT foi utilizado para Imaris interface com MATLAB. As intensidades de voxel foram trocadas emlocal coordena. Os pontos do espectro de cores codificadas (azul 0-5 pm, verdes, 6-10 uM, amarelas e vermelhas uM 11-15&gt; 15 uM) representam a forma de distância da membrana nuclear. Escala de 5 ^ m. <img alt="Figura 4" src="/files/ftp_upload/4233/4233fig4.jpg" /> Figura 4. Representação gráfica da distribuição distal dos pontos traçados no espectro de cores codificadas em intervalos de 5 uM é utilizada para visualizar a distância dos voxels da membrana nuclear. Nenhum tratamento e pré-tratamento com um antagonista (AS-30, 1 uM, 30 min) antes de tratamento com o agonista (CRF, 1 uM, 30 min) não mostram diferenças (ns) significativa na contracção GPCR. O tratamento das células com agonista (CRF, 1 uM, 30 min) reduz progressivamente a distância entre o número de CRF-R2 contendo voxels quando comparado com nenhum tratamento 0-5 ^ m (ns), 6-15 uM (** p &lt;0,01) e&gt; 15 mm (*** p &lt;0,005), ou AS-30 do tratamento 0-10 fim (ns), 11-15<html> uM (** p &lt;0,01) e&gt; 15 mm (*** p &lt;0,005). <img alt="Figura 5" src="/files/ftp_upload/4233/4233fig5.jpg" /> Figura 5. Limitação da análise morfométrica 2D de células HEK 293 transfectadas com HA-CRF-R2. A secção do plano médio de células (3-4μm acima da lamela de vidro) que mostra o centro dos núcleos visualizado com DAPI e HA-CRF-R2 sondadas com anti-HA e visualizados utilizando Alexa 488nm conjugado anti-ratinho (IgG 1) secundário anticorpo e adquiridas com CLS não mostra nenhuma diferença entre (A) sem tratamento (NT) e (B) agonista (CRF, 1 uM, 30 min), enquanto que os pontos de aderência dos receptores são muito diferentes. Filme 1. Livremente rotativo modelo 3D em &quot;Surpass&quot; modo de células HEK 293 transfectadas com HA-CRF-R2, nenhum tratamento, para avaliar as diferenças entre o fenótipo celular proteínas receptoras. Barra de escala de 5-20 ^ m./ Files/ftp_upload/4233/4233movie1.avi &quot;target =&quot; _blank &quot;&gt; Clique aqui para ver filme. Filme 2. Livremente rotativo modelo 3D em &quot;Surpass&quot; modo de células HEK 293 transfectadas com HA-CRF-R2, o tratamento com o agonista, CRF (CRF, 1 uM, 30 min) para avaliar diferenças entre os fenótipos celulares proteínas receptoras. Barra de escala 5-20 m. <a href="http://www.jove.com/files/ftp_upload/4233/4233movie2.avi" target="_blank">Clique aqui para ver filme.</a> Filme 3. Livremente rotativo 3D em &quot;Surpass&quot; modo de células HEK 293 transfectadas com HA-CRF-R2, o pré-tratamento com um antagonista (AS-30, 1 uM, 30 min) antes de tratamento com o agonista (CRF, 1 uM, 30 min ) para avaliar diferenças entre os fenótipos celulares proteínas receptoras. Barra de escala 5-20 m. <a href="http://www.jove.com/files/ftp_upload/4233/4233movie3.avi" target="_blank">Clique aqui para ver filme.</a>

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Nós mostramos que o tratamento CRF induz uma alteração significativa na morfologia e localização do CRF-R2. A mudança de CRF-R2 foi inibida pelo tratamento com o antagonista selectivo. Nós demonstramos que as modificações do receptor não foi detectada e não pode ser medida usando as técnicas padrão de 2D multiespectrais. A capacidade para estudar as imagens 3D complexas é essencial para incorporar a complexidade de parâmetros biológicos para análise morfométrica. Fomos capazes de fazer medições em 3D...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Rim Embrionário Humano (HEK293) </td><td> American Type Culture Collection </td><td> CRL-1573 </td><td></td></tr><tr><td> Dulbecco modificado por Eagle (DMEM) </td><td> Invitrogen </td><td> 11965118 </td><td></td></tr><tr><td> Soro fetal bovino (FBS) </td><td> Invitrogen </td><td> SH30070.03 </td><td></td></tr><tr><td> AlexaFluor-488 (IgG2b) </td><td> Invitrogen </td><td> A-11001 </td><td></td></tr><tr><td> monoclonal anti-HA.11 (IgG 1) </td><td> Covance </td><td> 16B12 </td><td></td></tr><tr><td> DAPI </td><td> Vector Laboratories </td><td> H-1200 </td><td>; </td></tr><tr><td> CRF </td><td> Sigma </td><td> C2917 </td><td></td></tr><tr><td> Antisauvagine-30 (AS-30) </td><td> Sigma </td><td> A4727 </td><td></td></tr></tbody></table>

an analytical tool that quantifies cellular morphology changes from three-dimensional fluorescence images

As ferramentas de software mais comuns de análise disponíveis para a medição de imagens de fluorescência são para dados bidimensionais (2D) que dependem de ajustes manuais para inclusão e exclusão de pontos de dados, e auxiliado por computador de reconhecimento de padrão para apoiar a interpretação e as conclusões da análise. Tornou-se cada vez mais importante ser capaz de medir imagens de fluorescência construídos a partir de conjuntos de dados tridimensionais (3D) a fim de ser capaz de captar a complexidade da dinâmica celular e compreender a base de plasticidade celular dentro de sistemas biológicos. Instrumentos sofisticados de microscopia permitiram a visualização de imagens 3D de fluorescência através da aquisição de imagens de fluorescência multiespectrais e software analítico poderoso que reconstrói as imagens a partir de pilhas confocal que, então, fornecem uma representação em 3D das imagens recolhidas 2D. Avançados métodos de projeto baseados em estereologia evoluíram a partir da aproximação e suposições da original estereologia modelo baseado em um mesmo em cortes de tecidos complexos 2. Apesar destes avanços científicos em microscopia, permanece a necessidade de um método automatizado analítico que explora plenamente os dados intrínsecos 3D para permitir a análise e quantificação das mudanças complexas na morfologia celular, localização de proteínas e tráfico receptor. Técnicas correntes disponíveis para quantificar as imagens de fluorescência incluem meta-Morph (Molecular Devices, Sunnyvale, CA) e Image J (NIH), que fornecem uma análise manual. Imaris software (Andor Technology, Belfast, Irlanda do Norte) fornece o recurso MeasurementPro, que permite a criação manual de pontos de medição que podem ser colocados em uma imagem de volume ou desenhados em uma série de fatias de 2D para criar um objeto 3D. Este método é útil para a medição de um único clique de ponto para medir a distância da linha entre dois objectos, ou para criar um polígono que inclui uma região de interesse, mas é difícil de aplicar a complex celulares estruturas de rede. Filamento Tracer (Andor) permite a detecção automática do neuronal 3D filamento como no entanto, este módulo foi desenvolvido para medir estruturas definidas, como neurônios, que são compostas de dendritos, axônios e espinhas (árvore-como estrutura). Este módulo foi engenhosamente utilizado para fazer medições morfológicas células não neuronais 3, no entanto, os dados de saída de informação de uma extensa rede celular por meio de um software que depende de uma forma da célula em vez de ser definido um modelo em forma amorfa celular. Para superar o problema da análise amorfo em forma de células e fazendo com que o software mais adequado para uma aplicação biológica, Imaris desenvolvido celular Imaris. Este foi um projeto científico com Eidgenössische Technische Hochschule, que tem sido desenvolvido para o cálculo da relação entre as células e organelas. Enquanto o software permite a detecção de restrições biológicas, forçando um núcleo por célulae usando membranas de células para as células do segmento, ele não pode ser utilizado para analisar os dados de fluorescência que não são idealmente contínuo porque ele cria na superfície celular, sem espaços vazios. Para nosso conhecimento, no momento nenhuma abordagem modificável pelo usuário automatizado que fornece informações morfométrica de 3D ​​imagens de fluorescência foi desenvolvido que atinge informação espacial celular de uma forma indefinida (Figura 1). Nós desenvolvemos uma plataforma analítica usando o módulo de software Imaris núcleo e Imaris XT interface com MATLAB (Mat Works, Inc.). Estas ferramentas permitem a medição 3D de células sem uma forma pré-definida e com os componentes da rede de fluorescência inconsistentes. Além disso, este método vai permitir aos pesquisadores que estenderam experiência em sistemas biológicos, mas não familiaridade com aplicativos de computador, para realizar a quantificação de alterações morfológicas na dinâmica celulares.

Watch this Scientific Journal Video about An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images at JoVE.com

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

Nós desenvolvemos uma plataforma de software que utiliza Imaris Neuroscience, ImarisXT e MATLAB para medir as alterações na morfologia de uma forma indefinida retirado tridimensional fluorescência confocal de células individuais. Esta nova abordagem pode ser utilizada para quantificar as alterações na forma da célula após a activação do receptor e, portanto, representa um instrumento adicional possível para a descoberta de drogas.

Uma ferramenta analítica que quantifica Alterações morfologia celular de imagens tridimensionais de fluorescência

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

an-analytical-tool-that-quantifies-cellular-morphology-changes-from

Research

JoVE Journal

Biology

14.5K visualizações.  University of California, San Francisco. Nós desenvolvemos uma plataforma de software que utiliza Imaris Neuroscience, ImarisXT e MATLAB para medir as alterações na morfologia de uma forma indefinida retirado tridimensional fluorescência confocal de células individuais. Esta nova abordagem pode ser utilizada para quantificar as alterações na forma da célula após a activação do receptor e, portanto, representa um instrumento adicional possível para a descoberta de drogas.

Vídeo: An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

Chip baseado em Cultura de Células tridimensional em perfundidos Micro-biorreatores

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Biologia

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Microfabricação de Chip porte Andaimes para tridimensional de cultivo celular

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

Um método de alto rendimento para estudar a morfologia global organela em S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Montagem, de carga, e alinhamento de uma célula Analytical ultracentrífuga Amostra

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

Alterações na morfologia da glândula mamária e risco de câncer de mama em ratos

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Isolamento de células epiteliais mamárias de tridimensional Mixed-célula esferoidal co-cultura

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Análise rápida e Exploração de microscopia de fluorescência Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET Imaging no hidrogéis tridimensionais

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear: Um método de compensação por imagem tridimensional do tecido adiposo tecido

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Abordagens quantitativas para o estudo de estruturas celulares e morfologia organelle em Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...