<html> Aqui vamos discutir os procedimentos experimentais que estão envolvidos no LUCAS [1-3]. Para ilustrar a prova de conceito de LUCAS vamos descrever o processo de imagem para uma amostra de sangue total. A. Imagem Set-up O LUCAS plataforma de imagem exibe vantagens significativas para fornecer uma alternativa econômica e compacta para existentes citometria de ponto de atendimento médico e ferramentas de diagnóstico, especialmente para ambientes com recursos limitados. Em vez de detectar a imagem das células, em vez LUCAS captura hologramas digitais das células que são criadas pela interferência da luz espalhada a partir de cada célula com a luz de fundo. Controle cuidadoso da coerência parcial espacial dos iluminação é fundamental para permitir a gravação holográfica. 1. Array de Sensor Digital A plataforma LUCAS utiliza um conjunto de sensores optoeletrônicos para gravar digitalmente hologramas célula individual. Para este fim, cobrado dispositivos casal (CCD; Modelos Amostra: KAI-11002, KAF-39000, da Kodak) ou complementary metal-oxide semiconductor-chips (CMOS, Modelo Amostra: MT9P031, Micron) pode ser usado. Tamanhos de pixel para a Kodak cobrado dispositivos casal, KAI-11002, KAF-39000, e os sensores de imagem CMOS Micron são 9 mM, 6,8 mM e 2,2 mM, com um FOV ativa de 10 cm2, 18 cm2, mm2 e 24,4, respectivamente. [1-2]. 2. Fonte de Luz Ao contrário da maioria das outras modalidades de imagem microscópica, LUCAS não requer um laser e, portanto, até mesmo um simples diodo emissor de luz (LED) pode ser usado para a iluminação. A fim de permitir a iluminação de comprimento de onda sintonizáveis, também podemos utilizar um monocromador com uma lâmpada Xenon (Cornerstone T260, Newport Corp), juntamente com um padrão de fibra de sílica fundida grade que consiste de um feixe de fibras (77564, Newport) e pinhole de ~ 100 mM de diâmetro localizado na ~ 5-10 cm acima da superfície do sensor. Esta configuração de iluminação ajustável de comprimento de onda fornece uma plataforma flexível onde as assinaturas holográfica das células pode ser ajustada, e híbridos assinaturas digitais podem ser sintetizados para melhorar a relação sinal-ruído para melhor caracterização precisão e especificidade. [3] B. Preparação da Amostra e Imagem A prova de conceito de LUCAS baseada no chip de imagem será demonstrado através de uma solução heterogênea, conforme descrito abaixo. Um protocolo semelhante poderia ser aplicada para vários outros tipos de células [1-3]. 1. Diluição de sangue total e preparação da solução heterogênea <ol><li> Para iniciar o processo, obter uma amostra de sangue total e microesferas de poliestireno de vários diâmetros (5, 10 e 20 mM. Duke Científica), e trazer todos eles para a temperatura ambiente. </li><li> Adicionar 2 mL de RPMI 1640 classic meios líquidos (Fisher Scientific) como diluente em um tubo de polipropileno estéril 5 mL. </li><li> Após incubar a amostra de sangue total por 30 minutos, pipetar até 10 L de amostra de eritrócitos sedimentados e transferi-lo para a solução de RPMI. </li><li> Adicionar um volume total de 20 microesferas de poliestireno mL em RPMI diluição dos eritrócitos, e agitar a solução da célula com cuidado pipetando ou usando agitador magnético com uma barra de agitação. </li><li> Coloque um volume total de 5-15 mL de solução heterogênea em uma tampa de deslizamento e colocar um segundo, lamínula idênticos sobre a solução usando uma pinça. A amostra deve ser imprensada entre as duas lamínulas uniformemente. </li><li> Em seguida, usando uma caneta de vácuo (NT57-636, Edmund Optics) de carga da amostra para a região ativa do conjunto de sensores de imagem. </li></ol> 2. Manchas de sangue total <ol><li> Começando com a preparação de uma mistura de 0,1% tamponado Eosina Y e Solução de metileno diluído New usando o poder tipo puro Eosina Y (MW = 691,85 Organics, Acros), zinco-free pura New corante azul de metileno (MW = 347,90 Organics, Acros), Monohydrate e oxalato de potássio (ACS Reagente 99,0%, Acros Organics), cada solução é purificado com um tamanho de 0,45 mM poros do filtro Syringless (Whatman) para a coloração de células brancas do sangue aquoso. </li><li> Então, mexa delicadamente um reagente coloração dissolvido aquosa com a amostra de sangue total em uma relação de volume de 1:1 em um copo de polipropileno, utilizando um agitador magnético por 2 minutos e incubar por 10 minutos. </li><li> Transferência de uma solução manchadas de sangue de 50 mL em um tubo de polipropileno 5 mL e diluir com solução de RPMI 1640 classic líquido (Fisher Scientific) de 1,95 mL para adquirir uma relação de volume de 1:200. Em seguida, vortex, a diluição por 30 segundos. A taxa de diluição diferentes também podem ser introduzidos nesta fase. </li><li> Pipetar uma solução célula corada de 10-100 mL entre lamínulas ou dentro de um reservatório microfluídicos. Em seguida, usando uma caneta de vácuo (NT57-636, Edmund Optics), coloque o espécime para a região ativa da matriz de sensor. </li></ol> RESULTADOS REPRESENTANTE ve_content &quot;&gt; <img src="/files/ftp_upload/1650/1650Fig1tmb.jpg" alt="Figura 1" border="0" /> Figura 1: LUCAS permite a contagem exata ea diferenciação digital de várias células e micro-objetos com base em suas assinaturas 2D holográfico sem o uso de lentes, lasers ou outros componentes ópticos volumosos. Assinaturas características LUCAS de vários micro-objetos são ilustrados nesta figura e são comparados com uma imagem de microscópio convencional obtida com uma lente objetiva de 40X. Por favor, <a href="https://www.jove.com/files/ftp_upload/1650/1650Fig1.jpg">clique aqui</a> para ver uma versão ampliada da figura 1. <img src="/files/ftp_upload/1650/1650Fig2.jpg" alt="Figura 2" /> Figura 2: (Esquerda) imagem Raw de uma mistura heterogênea contendo células vermelhas do sangue, 10um, 5um e 3 partículas um. (Direito) totalmente automatizado resultados da caracterização LUCAS para o mesmo campo de visão são ilustradas. Note que o algoritmo de decisão é robusto para caracterizar regiões de alta densidade, bem como partículas com baixo sinal-ruído, tais como as contas 3um. <img src="/files/ftp_upload/1650/1650Fig3tmb.jpg" alt="Figura 3" border="0" /> Figura 3: O LUCAS interface personalizada é ilustrado. Baseados em Java software LUCAS permite entradas para várias condições experimentais, tais como o tamanho do pixel do sensor ou o comprimento de onda da luz. Seleção de um campo específico de vista sobre a imagem também pode ser feita e os padrões de célula-alvo pode ser definido pelo usuário para construir uma biblioteca sombra estatística celular. A imagem adquirida LUCAS pode então ser caracterizado com base em dados de treinamento (ou seja, a biblioteca sombra de células) e da imagem marcada (contados) é exibida para o usuário. Estatísticas dos resultados contam também são armazenados como um arquivo XML para análise posterior.

<ol>
	<li>Seo, S., Su, T., Tseng, D. K., Erlinger, A., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Lensfree+Holographic+Imaging+for+On-Chip+Cytometry+and+Diagnostics".">"Lensfree Holographic Imaging for On-Chip Cytometry and Diagnostics".</a> Lab Chip. , (2009).</li><li>Su, T., Seo, S., Erlinger, A., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="High-throughput+Imaging+and+Characterization+of+a+Heterogeneous+Cell+Solution+on+a+Chip"+.">"High-throughput Imaging and Characterization of a Heterogeneous Cell Solution on a Chip" .</a> Biotechnology and Bioengineering. , (2008).</li><li>Su, T., Seo, S., Erlinger, A., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Multi-color+LUCAS:+Lensfree+on-chip+cytometry+using+tunable+monochromatic+illumination+and+digital+noise+reduction".">"Multi-color LUCAS: Lensfree on-chip cytometry using tunable monochromatic illumination and digital noise reduction".</a> Cellular and Molecular Bioengineering. , (2008).</li></ol>

Temos ilustrado que a plataforma LUCAS pode contar com precisão e identificar micro-objects/cells vários em um chip com base em suas assinaturas holográfico, e fornece uma ferramenta promissora para o ponto de atendimento diagnósticos médicos e de alto rendimento biologia celular. Com precisão, a fim de processar os padrões registrados holográfico, implementamos um software customizado decisão LUCAS. Este algoritmo, que utiliza um banco de dados de difração de imagem estatística criado por formação de imag...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Charged couple device (CCD)</td>
<td>&nbsp;</td>
<td>KODAK</td>
<td>KAI-11002</td>
<td>&nbsp;</td>
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<td>Charged couple device (CCD)</td>
<td>&nbsp;</td>
<td>KODAK</td>
<td>KAF-39000</td>
<td>&nbsp;</td>
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<td>Complementary Metal-Oxide-Semiconductor (CMOS)</td>
<td>&nbsp;</td>
<td>Micron</td>
<td>MT9P031</td>
<td>&nbsp;</td>
<tr>
<td>Xenon Lamp</td>
<td>&nbsp;</td>
<td>Newport Corp.</td>
<td>Cornerstone T260</td>
<td>&nbsp;</td>
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<td>Vacuum pen</td>
<td>&nbsp;</td>
<td>Edmund Optics</td>
<td>NT57-636</td>
<td>&nbsp;</td>
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<td>5, 10, and 20 &mu;m Microbeads</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>4000 Series</td>
<td>&nbsp;</td>
<tr>
<td>RPMI</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>1640</td>
<td>&nbsp;</td>
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<td>Pure Eosin Y</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>MW=691.85</td>
<td>&nbsp;</td>
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<td>New Methylene Blue(NMB) Dye</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>MW=347.90</td>
<td>&nbsp;</td>
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<td>Potassium Oxalate Monohydrate</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>99.0% Reagent ACS</td>
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lensless on-chip imaging of cells provides a new tool for high-throughput cell-biology and medical diagnostics

Óptico convencional microscópios células de imagem pelo uso de lentes objetivas que trabalham em conjunto com outras lentes e componentes ópticos. Embora bastante eficaz, esta abordagem clássica tem algumas limitações para a miniaturização da plataforma de imagem para torná-lo compatível com o estado avançado da arte em microfluídica. Neste relatório, nós apresentamos detalhes experimental de um conceito de imagem sem lente no chip denominado LUCAS (<strong<em&gt; L</em</strong&gt; Ensless<strong<em&gt; U</em</strong&gt; Ltra-wide campo de visão<strong<em&gt; C</em</strong&gt; Monitoramento ell<strong<em&gt; A</em</strong&gt; Rray plataforma baseada em<strong<em&gt; S</em</strong&gt; Hadow imagem) que não requer quaisquer objectivos microscópio ou outros componentes ópticos volumosos para a imagem de uma solução heterogênea de células sobre um campo ultra-wide de vista que pode abranger tão grande quanto ~ 18 centímetros<sup&gt; 2</sup&gt;. Além disso, ao contrário dos microscópios convencionais, LUCAS pode imagem uma solução celular heterogêneo de juros sobre uma profundidade de campo de ~ 5 mm sem a necessidade de recentrar o que corresponde a até ~ volume de amostra 9 mL. Esta plataforma de imagem registra a sombra (ou seja, sem lente hologramas digitais) de cada célula de interesse dentro do seu campo de visão e processamento digital automatizado destas sombras célula pode determinar o tipo, a contagem e as posições relativas de células dentro da solução. Porque não exige quaisquer componentes volumosos óptico ou mecânico estágios a verificação, ele oferece uma plataforma significativamente miniaturizados que, ao mesmo tempo reduz o custo, que é muito importante para ponto de atendimento, especialmente as ferramentas de diagnóstico. Além disso, a taxa de transferência de imagem desta plataforma é ordens de magnitude melhor do que microscópios ópticos convencionais, o que poderia ser extremamente valioso para high-throughput biologia celular-experimentos.

Watch this Scientific Journal Video about Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics at JoVE.com

Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics

Lensfree imagens on-chip e caracterização de células é ilustrada. Esta abordagem on-chip imagens de células fornece uma ferramenta compacta e cost-effective para diagnósticos médicos e de alto rendimento aplicações de biologia celular, tornando-o especialmente adequado para locais de poucos recursos.

Imagem On-chip sem lente de Células fornece uma nova ferramenta de alta capacidade: Biologia Celular e Diagnóstico Médico

Conventional optical microscopes image cells by use of objective lenses that work together with other lenses and optical components. While quite ...

lensless-on-chip-imaging-cells-provides-new-tool-for-high-throughput

Research

JoVE Journal

Biology

11.9K visualizações.  University of California, Los Angeles. Lensfree imagens on-chip e caracterização de células é ilustrada. Esta abordagem on-chip imagens de células fornece uma ferramenta compacta e cost-effective para diagnósticos médicos e de alto rendimento aplicações de biologia celular, tornando-o especialmente adequado para locais de poucos recursos.

Vídeo: Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics

Tracheotomy: A Method for Transplantation of Stem Cells to the Lung

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell therapy may alleviate many of the symptoms associated with lung disease and induce alveolar repair in adults. Concurrent with the ongoing search for stem cells applicable for human treatment, precise delivery and homing (to the site of disease) must be reassured for successful therapy. Here, I report that stem cells can safely be instilled via the trachea opening a non-stop route to the lung. This method involves a skin incision, caudal insertion of a cannula into and along the tracheal lumen, and injection of a stem cell vehicle mixture into airways of the lung. A broad range of media solutions and stabilizers can be instilled via tracheotomy, resulting in the ability to deliver a wider range of cell types. With alveolar epithelium confining these cells to the lumen, lung expansion and negative pressure during inhalation may also assist in stem cell integration. Tracheal delivery of stem cells, with a quick uptake and the ability to handle a large range of treatments, could accelerate the development of cell-based therapies, opening new avenues for treatment of lung disease.

Significant breakthroughs in stem cell identification are continuously being made. To translate these discoveries, however, novel methods for cellular delivery must be devised. Here I report that the airways provide a safe route for stem cell transplantation to the lungs.

Traqueotomia: Um Método para Transplante de Células-Tronco para o pulmão

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell ...

Biologia

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

Um dispositivo de geração de Gradient microfluídicos de Biologia Celular

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

Bioquímica e Alto Rendimento Avaliação microscópica de massa gorda em<em&gt; Caenorhabditis elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Anti-Nuclear Antibody Screening Using HEp-2 Cells

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although IIF is an excellent screening test in expert hands, the technical difficulties of processing and reading IIF slides &#8211; such as the labor intensive slide processing, manual reading, the need for experienced, trained technologists and the use of dark room &#8211; make the IIF method difficult to fit in the workflow of modern, automated laboratories.
The first and crucial step towards high quality ANA screening is careful slide processing. This procedure is labor intensive, and requires full understanding of the process, as well as attention to details and experience.
Slide reading is performed by fluorescent microscopy in dark rooms, and is done by trained technologists who are familiar with the various patterns, in the context of cell cycle and the morphology of interphase and dividing cells. Provided that IIF is the first line screening tool for SARD, understanding the steps to correctly perform this technique is critical.
Recently, digital imaging systems have been developed for the automated reading of IIF slides. These systems, such as the NOVA View Automated Fluorescent Microscope, are designed to streamline the routine IIF workflow. NOVA View acquires and stores high resolution digital images of the wells, thereby separating image acquisition from interpretation; images are viewed an interpreted on high resolution computer monitors. It stores images for future reference and supports the operator&#8217;s interpretation by providing fluorescent light intensity data on the images. It also preliminarily categorizes results as positive or negative, and provides pattern recognition for positive samples. In summary, it eliminates the need for darkroom, and automates and streamlines the IIF reading/interpretation workflow. Most importantly, it increases consistency between readers and readings. Moreover, with the use of barcoded slides, transcription errors are eliminated by providing sample traceability and positive patient identification. This results in increased patient data integrity and safety.
The overall goal of this video is to demonstrate the IIF procedure, including slide processing, identification of common IIF patterns, and the introduction of new advancements to simplify and harmonize this technique.

Indirect immunofluorescent (IIF) assays have traditionally been used for the detection of antinuclear antibodies (ANA) in human serum. The presence of these antibodies can aid in the diagnosis of systemic autoimmune rheumatic diseases (SARD). This protocol demonstrates how to effectively perform the IIF technique to accurately detect these autoantibodies.

Anti-Nuclear Antibody Triagem Usando Células HEp-2

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

De alta capacidade Análise de Imagem de esferóides tumorais: uma aplicação de software de fácil utilização para medir o tamanho de esferóides automaticamente e com precisão

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Rastreio de alto rendimento para moduladores químicos de Genes Post-transcriptionally Regulamentados

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

High-Throughput Metabolic Profiling for Model Refinements of Microalgae

Metabolic models are reconstructed based on an organism&#39;s available genome annotation and provide predictive tools to study metabolic processes at a systems-level. Genome-scale metabolic models may include gaps as well as reactions that are unverified experimentally. Reconstructed models of newly isolated microalgal species will result in weaknesses due to these gaps, as there is usually sparse biochemical evidence available for the metabolism of such isolates. The phenotype microarray (PM) technology is an effective, high-throughput method that functionally determines cellular metabolic activities&#160;in response to a wide array of entry metabolites. Combining the high throughput phenotypic assays with metabolic modeling can allow existing metabolic network models to be rapidly reconstructed or optimized by providing biochemical evidence to support and expand genomic evidence. This work will show the use of PM assays for the study of microalgae by using the green microalgal model species Chlamydomonas reinhardtii as an example. Experimental evidence for over 254 reactions obtained by PM was used in this study to expand and refine a genome-scale C. reinhardtii metabolic network model, iRC1080, by approximately 25 percent. The protocol created here can be used as a basis for functionally profiling the metabolism of other microalgae, including known microalgae mutants and new isolates.

This protocol demonstrates the use of a phenotype microarray (PM) technology platform to define metabolic requirements of Chlamydomonas reinhardtii, a green microalga, and refine an existing metabolic network model.

Perfil metabólico de alta produtividade para refinamentos de modelo de microalgas

Metabolic models are reconstructed based on an organism&#39;s available genome annotation and provide predictive tools to study metabolic processes at a ...

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental inputs into stress tolerance, pathogenicity, and other key components of fitness. This manuscript describes a microscope-based assay that tracks approximately 105&#160;Saccharomyces cerevisiae microcolonies per experiment. After automated time-lapse imaging of yeast immobilized in a multiwell plate, microcolony growth rates are easily analyzed with custom image-analysis software. For each microcolony, expression and localization of fluorescent proteins and survival of acute stress can also be monitored. This assay allows precise estimation of strains&#39; average growth rates, as well as comprehensive measurement of heterogeneity in growth, gene expression, and stress tolerance within clonal populations.

Yeast growth phenotypes are precisely measured through highly parallel time-lapse imaging of immobilized cells growing into microcolonies. Simultaneously, stress tolerance, protein expression, and protein localization can be monitored, generating integrated datasets to study how environmental and genetic differences, as well as gene-expression heterogeneity among isogenic cells, modulate growth.

Imagem ao vivo de alta taxa de microcolônias para medir heterogeneidade no crescimento e expressão genética

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental ...

A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell function, and therefore DNA damage may play a critical role in the development of, conceivably, all major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging. Single-cell gel electrophoresis (i.e., the comet assay) is one of the most common and sensitive methods to study the formation and repair of a wide range of types of DNA damage (e.g., single- and double-strand breaks, alkali-labile sites, DNA-DNA crosslinks, and, in combination with certain repair enzymes, oxidized purines, and pyrimidines), in both in vitro and in vivo systems. However, the low sample throughput of the conventional assay and laborious sample workup are limiting factors to its widest possible application. With the "scoring" of comets increasingly automated, the limitation is now the ability to process significant numbers of comet slides. Here, a high-throughput (HTP) variant of the comet assay (HTP comet assay) has been developed, which significantly increases the number of samples analyzed, decreases assay run time, the number of individual slide manipulations, reagent requirements, and risk of physical damage to the gels. Furthermore, the footprint of the electrophoresis tank is significantly decreased due to the vertical orientation of the slides and integral cooling. Also reported here is a novel approach to chilling comet assay slides, which conveniently and efficiently facilitates the solidification of the comet gels. Here, the application of these devices to representative comet assay methods has been described. These simple innovations greatly support the use of the comet assay and its application to areas of study such as exposure biology, ecotoxicology, biomonitoring, toxicity screening/testing, together with understanding pathogenesis.

The comet assay is a popular means of detecting DNA damage. This study describes an approach to running slides in representative variants of the comet assay. This approach significantly increased the number of samples while decreasing assay run-time, the number of slide manipulations, and the risk of damage to gels.

Uma abordagem de ensaio de cometa de alto rendimento para avaliar danos ao DNA celular

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell ...

A High-Throughput Platform for Culture and 3D Imaging of Organoids

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by standard imaging approaches. This protocol describes a way to prepare microfabricated organoid culture chips, which enable multiscale, 3D live imaging on a user-friendly instrument requiring minimal manipulations and capable of up to 300 organoids/h imaging throughput. These culture chips are compatible with both air and immersion objectives (air, water, oil, and silicone) and a wide range of common microscopes (e.g., spinning disk, point scanner confocal, wide field, and brightfield). Moreover, they can be used with light-sheet modalities such as the single-objective, single-plane illumination microscopy (SPIM) technology (soSPIM). 
The protocol described here gives detailed steps for the preparation of the microfabricated culture chips and the culture and staining of organoids. Only a short length of time is required to become familiar with, and consumables and equipment can be easily found in normal biolabs. Here, the 3D imaging capabilities will be demonstrated only with commercial standard microscopes (e.g., spinning disk for 3D reconstruction and wide field microscopy for routine monitoring).

This paper presents a fabrication protocol for a new type of culture substrate with hundreds of microcontainers per mm2, in which organoids can be cultured and observed using high-resolution microscopy. The cell seeding and immunostaining protocols are also detailed.

Uma plataforma de alto rendimento para cultura e imagem 3D de organoides

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by ...