Os autores gostariam de agradecer ao Dr. Stephen Kingsmore e do Centro de Medicina Pediátrica Genoma em hospitais pediátricos Mercy e clínicas para o uso de clusters de computação para a nossa análise de dados, a equipe de Illumina serviço de campo (Elizabeth Boyer, Scott Cook e Mark Cook) e técnico equipa de consultores para as suas respostas rápidas e sugestões úteis sobre o funcionamento do instrumento de próxima geração de sequenciamento de DNA, HiScanSQ e análise de dados de qualidade. Este trabalho foi financiado em parte pelo National Institutes of Health Grant HL080042 (para SQY) e start-up fundo e dotação de hospitais infantis de Misericórdia e Clínicas, da Universidade de Missouri, em Kansas City (para SQY).

<html> Um fluxograma descrevendo este protocolo é mostrado na Figura 1. 1. O tratamento de células com trombina, RNA Isolation, Quality Assessment e Quantificação de RNA <ol><li> Cultura As células endoteliais microvasculares de pulmão humano (HMVEC-LBL) a 90-100% de confluência em placas de 6 poços em meio EGM-2 com 5% de FBS, factores de crescimento e antibióticos (Lonza, cat # CC-3202). </li><li> Alterar media para os meios de comunicação de fome (0% de FBS) 30 min antes do tratamento com trombina. </li><li> Tratar as células com 0,05 U / ml de trombina ou deixar sem tratamento como um controlo durante 6 horas a 37 ° C e 5% CO 2. </li><li> Isolar o RNA total a partir das células tratadas e de controlo, usando o Ambion mir Vana kit de acordo com as instruções do fabricante. </li><li> Avaliar a qualidade do RNA com uma Eukaryotic StdSense Experion chip de RNA de acordo com o protocolo padrão na Estação Electrophoresis Experion (Automated= &quot;_blank&quot;&gt; Www.bio-rad.com). </li><li> Quantificar o ARN utilizando um método padrão espectrofotométrico. </li></ol> 2. Construção de biblioteca e Sequenciamento <ol><li> Use de 1 ug de ARN total de elevada qualidade por exemplo como material de partida. </li><li> Para construir a biblioteca, seguir o procedimento padrão da Illumina (protocolo n º 15008136 Rev. A). Neste protocolo, duas rodadas de poli (A) de ARNm contendo selecções são realizadas para remover rRNA para minimizar a sequenciação de rRNA. </li><li> Avaliar a qualidade das bibliotecas utilizando um chip de ADN Experion 1K de acordo com o protocolo padrão na Estação Electrophoresis Experion automatizada ( <a href="http://www.bio-rad.com" target="_blank">www.bio-rad.com</a> ). </li><li> Quantificar a biblioteca utilizando qPCR: Usar uma biblioteca que tenha sido previamente sequenciados como uma curva padrão e iniciadores específicos para os adaptadores ligados. Utilizar uma gama de diluições das bibliotecas desconhecidas (por exemplo, 1:100, 1:500 e 1:1000). Execute o acco qPCRrding com o protocolo MM SyberGreen e calcular a concentração original de estoque de cada biblioteca. </li><li> Diluir os stocks de biblioteca de 10 nM e armazenar a -20 ° C até estar pronto para agrupar uma célula de fluxo. </li><li> Quando estiver pronto para agrupar uma célula de fluxo, descongelar a placa reagente CBOT num banho de água. CBOT é um instrumento utilizado Illumina para agilizar o processo de geração de cluster. </li><li> Lavar o instrumento CBOT. </li><li> Desnaturar as bibliotecas: Combinar 13 ul de 1x TE e 6 uM biblioteca ul 10 e, para o lado do tubo, adicionar 1 ml de NaOH 1 N (fornecido pela Illumina). Vortex, girada para baixo, incubar à temperatura ambiente durante 5 min e colocar as bibliotecas desnaturados em gelo. </li><li> Diluir as bibliotecas: Diluir as bibliotecas desnaturados com pré-gelada de tampão de hibridação (HT1, fornecido pela Illumina) através da combinação de 996 ul HT1 e 4 uL biblioteca desnaturado para uma concentração final de 12 pM. Coloque as bibliotecas desnaturados e diluído em gelo. </li><li> Inverter cada fila de tubos da placa CBOT,assegurar que todos os reagentes são descongeladas. Girar a placa, remover / punção os selos tiras e coloque sobre a CBOT. </li><li> Ul alíquota 120 das bibliotecas diluídas, desnaturados para um tubo de tira, rotulado 1-8. Adicionar 1,2 uL diluído, biblioteca de controle PhiX desnaturado (de Illumina) em cada tubo como uma espiga de controle. Vortex e girar os tubos e carregá-los na CBOT na orientação correta (tubo n º 1 para a direita). </li><li> Carregar uma célula de fluxo e colector para a CBOT. </li><li> Completar o fluxo de verificar e começar a corrida de agrupamento. </li><li> Após a corrida é completa, veja a entrega de reagente em todas as pistas. Tome nota de quaisquer anormalidades. Quer iniciar a execução de sequenciação imediatamente ou armazenar a célula de fluxo no tubo desde a 4 ° C. </li><li> Descongele o seqüenciamento por síntese (SBS, Illumina) reagentes. </li><li> Coloque os reagentes para os pontos apropriados nas bandejas de reagentes, tomando cuidado para não tocar os outros reagentes depois de tocar o mix de clivagem. </li><li> Usando um nãon-seqüenciamento célula de fluxo (isto é, que foi sequenciado anteriormente), principais linhas de reagente duas vezes. </li><li> Limpar completamente a célula de fluxo de sequenciação com EtOH a 70% e Kimwipes, seguido de EtOH a 70% e de papel de lente. Inspecione a célula de fluxo para qualquer fiapo. Re-limpá-lo, se necessário. </li><li> Carregar a célula de fluxo para o sequenciador e executar um fluxo de verificação para assegurar que a vedação entre os tubos de distribuição e a célula de fluxo é apertado. </li><li> Iniciar a execução seqüenciamento. </li><li> Avaliar as métricas de qualidade (por exemplo, a densidade de cluster, clusters de passar filtro, Q30, intensidade), como eles se tornam disponíveis durante a corrida. </li><li> Monitorar a intensidade em toda a corrida. </li><li> Após 101 ciclos são concluídos, realizar a química de retorno para completar a segunda leitura: Descongelar os reagentes finais emparelhadas e o buffer de Incorporação segunda leitura (ICB, um componente do SBS reagentes, Illumina) e carregar os reagentes. </li><li> Continuar a corrida seqüenciamento, avaliando 2 ª leitura intensidade, um Q30nd outras métricas de qualidade como os avanços de execução. </li></ol> 3. Análise de Dados <ol><li> Use a versão mais recente do casava (Illumina, atualmente 1.8.2) para converter os arquivos de base de chamada (. Bcl) arquivos em arquivos. Fastq, estabelecendo fastq-cluster contagem a 0 para garantir a criação de um arquivo fastq único para cada amostra . Descompactar os arquivos fastq para análise a jusante. </li><li> Realizar o alinhamento final emparelhado utilizando as mais recentes versões do TopHat (1.4.1) 5, que alinha RNA-seq lê para mamíferos de tamanho genomas usando o ultra high-throughput alinhador de leitura curta (Bowtie, 0.12.7) 6 e SAMtools (0,1. 17) 7. SAMtools implementa vários utilitários para pós-processamento de alinhamentos no formato SAM. O transcriptoma de referência humano pode ser baixado do iGenomes ( <a href="http://www.illumina.com" target="_blank">www.illumina.com</a> ). Na corrida TopHat, usamos todas as configurações padrão de parâmetros, incluindo a opção de tipo de biblioteca como fr-unstranded (padrão). </li><li> Nosção a CuffDiff programa, uma parte dos botões de punho (1.3.0) 8 pacote de software, comparar as células tratadas com trombina para as células de controlo de filtrar os transcritos de genes expressos diferencialmente na antiga com base na referência do transcriptoma humano. Esta comparação detectar a expressão diferencial de transcritos conhecidos. Use o Microsoft Excel para visualizar o resultado em forma de tabela. Na execução do programa Abotoaduras, usamos todos os parâmetros predefinidos. Essas transcrições de genes com FPKM &lt;0,05 e p&gt; 0,05 são filtrados. </li><li> Para detectar novas isoformas, executar Cufflinks sem transcriptoma de referência. Comparar os arquivos de transcritos de amostras de referência, utilizando o genoma Cuffcompare e testar a expressão diferencial com Cuffdiff usando os arquivos de transcrição combinados trombina como o genoma de referência para uma análise e os ficheiros de controlo de transcrição combinados, como no genoma de referência para uma segunda análise. Use o Microsoft Excel para visualizar o resultado em formato tabular. Novamente, thostranscrições de e genes com FPKM &lt;0,05 e p&gt; 0,05 são filtrados. Após esta etapa, os pesquisadores podem optar por carregar uma lista de transcrições de notificação recente para o site UCSC Genome Browser ( <a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a> ) para verificar a sua validade por uma inspeção manual. </li><li> Apresentar listas de genes diferencialmente expressos para Análise Ingenuity Pathway (IPA, <a href="www.ingenuity.com" target="_blank">www.ingenuity.com</a> ) para a caracterização dos genes e as vias afetadas pelo tratamento trombina. Nesta etapa, os pesquisadores podem optar por usar cummerbund ( <a href="http://compbio.mit.edu/cummeRbund/" target="_blank">http://compbio.mit.edu/cummeRbund/</a> ), um pacote de R que é projetado para ajudar e simplificar a tarefa de analisar Cufflinks saída de RNA-seq, para ajudar a gerenciar, visualizar e integrar todos os dados produzidos por uma análise Cuffdiff. </li></ol> 4. Validação dos resultados RNA-seq por quantitativa em tempo real-PoReacção lymerase Chain (qRT-PCR) <ol><li> Realizar isolamento de RNA total do controle e da trombina tratados HMVEC Lbl células, RNA qualidade de avaliação e quantificação de RNA descrito nos Passos 1,4-1,6. </li><li> Gerar DNA complementar a partir de 1 ug de RNA total de cada amostra com o Superscript First-Strand III Síntese Kits sistema RT, seguindo as instruções do fabricante (Invitrogen, 18080-051). </li><li> Executar qRT-PCR em uma VIIa Applied Biosystems 7 Real-Time PCR System usando Taqman Assay-on-Demand oligonucleótidos concebidos para a detecção de CUGBP, Elav-likefamilymember1 (CELF1, Hs00198069_m1), Fanconianemia, complementationgroupD2 (FANDCD2, Hs00276992_m1), TNFreceptor -associated factor 1 (TRAF1, Hs01090170_m1) e β-actina (ACTB, Hs99999903_m1). Cada amostra tinha um modelo equivalente a 5 ng de RNA total. Medir a quantificação utilizando o método DDCT e normalizar a β-actina. Cada ensaio foi realizado através de, pelo menos, três repetições biológica. </li></ol>

<ol>
	<li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nat. Rev. Genet. 10, 57-63 (2009).</li><li>Shendure, J., Ji, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+DNA+sequencing.">Next-generation DNA sequencing.</a> Nat. Biotechnol. 26, 1135-1145 (2008).</li><li>Marioni, J. C., Mason, C. E., Mane, S. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopHat:+discovering+splice+junctions+with+RNA-Seq.">TopHat: discovering splice junctions with RNA-Seq.</a> Bioinformatics. 25, 1105-1111 (2009).</li><li>Langmead, B., Trapnell, C., Pop, M., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+and+memory-efficient+alignment+of+short+DNA+sequences+to+the+human+genome.">Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.</a> Genome Biol. 10, R25 (2009).</li><li>Li, H., 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+Sequence+Alignment/Map+format+and+SAMtools.">The Sequence Alignment/Map format and SAMtools.</a> Bioinformatics. 25, 2078-2079 (2009).</li><li>Trapnell, C., 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=Transcript+assembly+and+quantification+by+RNA-Seq+reveals+unannotated+transcripts+and+isoform+switching+during+cell+differentiation.">Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation.</a> Nat. 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As principais etapas Manipulação de RNA: RNases irá degradar ainda os mais elevada qualidade de ARN, por isso deve ser tomado cuidado durante o isolamento, o armazenamento e utilização de RNA 10. Luvas são sempre usadas para evitar a contaminação por RNases encontrados em mãos humanas. As luvas devem ser mudados frequentemente, especialmente depois de tocar a pele, maçanetas ou outras superfícies comuns. Um conjunto de pipetas deve ser dedicado exclusivame...

<table border="1"><tbody><tr><td width="183"> Reagentes ou Equipamentos </td><td width="85"> Companhia </td><td width="185"> Número de catálogo </td><td width="359"> Comentários </td></tr><tr><td> As células endoteliais microvasculares de pulmão humano </td><td> Lonza </td><td> CC-2815 </td><td></td></tr><tr><td> Lonza, Kit Bala </td><td> Lonza </td><td> CC-3202 </td><td> Contém EGM-2, FBS, factores de crescimento e antibióticos </td></tr><tr><td> Trombina </td><td> Sigma </td><td> T4393 </td><td></td></tr><tr><td> Ambion mir Vana Kit </td><td> Life Technologies </td><td> AM 1560 </td><td></td></tr><tr><td> RNase-Zap </td><td> Life Technologies </td><td> AM9782 </td><td></td></tr><tr><td> Experion StdSens RNA </td><td> Bio-Rad </td><td> 700-7103 </td><td></td></tr><tr><td> TruSeq RNA PreparaçãoConjunto </td><td> Illumina </td><td> FC-122-1001 </td><td></td></tr><tr><td> Contas AMPureXP </td><td> Beckman Coulter </td><td> A63881 </td><td></td></tr><tr><td> Sobrescrito transcriptase reversa II </td><td> Life Technologies </td><td> 18064-014 </td><td></td></tr><tr><td> Experion 1K DNA </td><td> Bio-Rad </td><td> 700-7107 </td><td></td></tr><tr><td> QuantiTect SyberGreen </td><td> Qiagen </td><td> 204163 </td><td></td></tr><tr><td> PE Cluster Geração Kit </td><td> Illumina </td><td> PE-401-3001 </td><td></td></tr><tr><td> PhiX Controle Kit </td><td> Illumina </td><td> FC110-301 </td><td></td></tr><tr><td> 200 Ciclo SBS Kit </td><td> Illumina </td><td> FC-401-3001 </td><td></td></tr><tr><td> HiScanSQ * </td><td> Illumina </td><td> SY-103-2001 </td><td></td></tr><tr><td> CBOT </td><td> Illumina </td><td> SY-301-2002 </td><td></td></tr><tr><td> qPCR máquina - Viia7 </td><td> Life Technologies </td><td> Equipamento Model # VIIA7 / # 10631261 </td><td> Ou equivalente </td></tr><tr><td> Experion Sistema </td><td> Bio Rad </td><td> 7007001 </td><td> Bioanalyzer é um sistema alternativo </td></tr><tr><td> Espectrofotômetro </td><td> Bio-Tek </td><td> Epoch Microplaca Espectrofotômetro </td><td> Ou equivalente </td></tr><tr><td> Centrífuga - Sorvall Legend XTR </td><td> Thermo Scientific </td><td> 75004521 </td><td> Ou equivalente </td></tr><tr><td> Suporte magnético </td><td> Life Technologies </td><td> AM10027 </td><td></td></tr><tr><td> 96 poços termociclador </td><td> Geral Fornecedor Lab </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Lista Tabela 3. Dos Reagentes chave e Mi Maiorquipment. *, No vídeo, em vez de HiSeq1000 HiScanSQ foi demonstrada. </td></tr></tbody></table>

rna-seq analysis of transcriptomes in thrombin-treated and control human pulmonary microvascular endothelial cells

A caracterização da expressão do gene em células por meio de medição de níveis de mRNA é uma ferramenta útil na determinação de como a maquinaria transcricional da célula é afectada por sinais externos (por exemplo, tratamento com drogas), ou como células diferem entre um estado saudável e de um estado de doença. Com o advento e aperfeiçoamento contínuo da próxima geração de tecnologias de sequenciação de DNA, RNA-sequenciação (RNA-seq) tornou-se um método cada vez mais popular de análise do transcriptoma para catalogar todas as espécies de transcritos, para determinar a estrutura da transcrição de todos os genes expressos e quantificar os níveis de expressão de mudar o conjunto total de transcritos de 1,2 um tecido, célula ou organismo determinado. RNA-seq está a substituir gradualmente microarranjos de DNA como um método preferido para a análise do transcriptoma porque tem as vantagens de um perfil transcriptoma completa, proporcionando um dado tipo digital (número de cópias de qualquer transcrição) e não se basear em qualquer sequen genómico conhecidoce 3. Aqui, apresentamos um protocolo completo e detalhado para aplicar RNA-seq para transcriptomas perfil em humanos pulmonares células endoteliais microvasculares com ou sem tratamento de trombina. Este protocolo é baseado em nosso estudo recente publicado intitulado &quot;RNA-seq revela Transcriptoma Novel de genes e suas isoformas em humanos pulmonares células endoteliais microvasculares Tratados com trombina,&quot; 4 em que realizaram com sucesso a primeira análise completa do transcriptoma humano pulmonares células endoteliais microvasculares tratado com trombina usando RNA-seq. Ele rendeu recursos sem precedentes para a experimentação ainda mais para obter insights sobre os mecanismos moleculares subjacentes trombina mediada disfunção endotelial na patogênese de doenças inflamatórias, câncer, diabetes e doença cardíaca coronária, e fornece novas pistas para potenciais alvos terapêuticos para essas doenças. O texto descritivo deste protocoloé dividido em quatro partes. A primeira parte descreve o tratamento de humanos pulmonares células endoteliais microvasculares com trombina e isolamento de ARN, análise de qualidade e de quantificação. A segunda parte descreve a construção da biblioteca e seqüenciamento. A terceira parte descreve a análise de dados. A quarta parte descreve um ensaio de RT-PCR de validação. Os resultados representativos de vários passos-chave são exibidos. Dicas úteis ou precauções para impulsionar o sucesso em passos-chave são fornecidos na seção de Discussão. Embora este protocolo utiliza humanas pulmonares células endoteliais microvasculares tratados com trombina, pode ser generalizado para transcriptomas perfil em ambas as células de mamíferos e não mamíferos e dos tecidos tratados com diferentes estímulos ou inibidores, ou para comparar transcriptomas em células ou tecidos entre um estado saudável e um estado de doença.

Para o Passo 1: O 28s: 18s razão é tradicionalmente utilizada como um indicador da degradação de ARN. Idealmente, o pico 28s deverá ter aproximadamente duas vezes a área da banda de 18s (uma proporção de 2), no entanto este rácio ideal não é muitas vezes visto na prática. Além disso, 28s: 18s rácios obtidos a partir de métodos espectrofotométricos pode subestimar a quantidade de degradação do RNA. Para quantificar mais precisamente a degradação, e, por conseguinte, a qualidade da amo...

Watch this Scientific Journal Video about RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells at JoVE.com

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

Este protocolo apresenta um procedimento completa e detalhada para aplicar RNA-seq, uma tecnologia de DNA poderoso próxima geração de sequenciação, de transcriptomas de perfil em humanos pulmonares células endoteliais microvasculares, com ou sem tratamento com trombina. Este protocolo é generalizável a várias células ou tecidos afetados por reagentes ou estados diferentes doenças.

RNA-seq Análise de transcriptomas de trombina-tratados e controle humano pulmonar células endoteliais microvasculares

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

rna-seq-analysis-transcriptomes-thrombin-treated-control-human

Research

JoVE Journal

Biology

21.8K visualizações.  Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City. Este protocolo apresenta um procedimento completa e detalhada para aplicar RNA-seq, uma tecnologia de DNA poderoso próxima geração de sequenciação, de transcriptomas de perfil em humanos pulmonares células endoteliais microvasculares, com ou sem tratamento com trombina. Este protocolo é generalizável a várias células ou tecidos afetados por reagentes ou estados diferentes doenças.

Vídeo: RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

Isolamento de células endoteliais da veia umbilical humana (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

Biologia

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolamento e cultura de células endoteliais pulmonares de camundongos Neonatal

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

Bioengenharia Humanos Networks microvascular em camundongos imunodeficientes

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

A cultura de células epiteliais nasais Humano na interface ar líquido

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

Isolamento de tubos endoteliais microvasculares de Artérias Rato Resistência

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

Análise do estresse oxidativo em embriões Zebrafish

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

O isolamento de células endoteliais de Murino Válvula

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

Face Detection En de óxido nítrico e superóxido na camada endotelial das artérias intactas

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

De alta resolução de imagem Time-lapse e análise automatizada de microtúbulos Dynamics em Viver umbilical humano Células Endoteliais da Veia

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.