Pesquisa tem sido apoiada pela Coulter H. Wallace Foundation Prémio Carreira No início de Pesquisa Translacional. Graças a Tim Andrews, Pediátrica Hematologia / Oncologia, para a ajuda com a coleta de amostra de sangue. Também graças ao Dr. Sam Wellhausen, Core Citometria de Fluxo, James Graham Brown Cancer Center, para ajudar com o funcionamento de amostras de citometria de fluxo.

Parte 1. Fabricação de dispositivos microfluídicos Um molde mestre de silício do dispositivo lise microfluídicos foi criado usando técnicas de fotolitografia com o equipamento na Universidade de Louisville salas limpas [1]. AutoCAD (Autodesk, Inc., San Rafael, CA) foi utilizado para gerar uma máscara de transparência (Fineline Imaging, Colorado Springs, CO) para criar réplicas negativas dos canais. O dispositivo foi fabricado lise microfluídica do molde mestre de silício usando técnicas litográficas suaves com o elastômero poli (dimetilsiloxano) (PDMS; Dow Corning, Midland, MI). O elastômero com os canais replicado foi lançado, e orifícios de acesso do canal foi perfurado com uma agulha de calibre 20 (peças pequenas, Miramar, FL.). O wafer PDMS foi irreversivelmente ligado a uma lâmina de vidro por meio de plasma de oxigênio e testado para a experimentação. Parte 2. Protocolo para a execução de dispositivo de lise Após o dispositivo micro foi fabricado e testado, experimentos com a lise de eritrócitos está pronto para começar. A seção seguinte detalha o protocolo para o dispositivo microfluídicos. 2,1 de Coleta de Amostras <ol><li> Coletar quatro mililitros de amostra de sangue da veia cubital mediana, na veia do antebraço anterior do paciente com heparina como anticoagulante em dois 2 ml verde vaccutainers topo. </li><li> Descartar tubo ml 2 primeiros, pois contém desalojado madura células endoteliais (falsos positivos). </li><li> Ressuspender o segundo tubo para misturar heparina com sangue girando para cima e para baixo; salvar imediatamente em gelo. </li><li> Transferir 1 ml de sangue para um tubo Eppendorf 1,5 ml. Processo a amostra de sangue imediatamente (dentro de uma hora da coleta). </li></ol> 2,2 dispositivo Prime microfluídicos com PBS <ol><li> Obter um dispositivo micro ligado ao vidro. Pressione-fit tubo de acesso (peças pequenas, Miramar, FL) de diâmetro ligeiramente maior nas entradas e saída (Figura 1). </li><li> Coloque uma agulha de seringa de calibre 30 (peças pequenas, Miramar, FL) para o tubo em cada uma das entradas, proporcionando a macro para fazer a interface micro. </li><li> Encha uma seringa de 1 ml com PBS 1X e certifique-se de se livrar de bolhas sacudindo a agulha da seringa. Conecte o PBS-carregados seringa de 1 ml para uma entrada no dispositivo microfluídicos. Empurre a seringa contendo o PBS 1X suavemente com a mão até que a solução sai de entrada 2, e logo em seguida braçadeira de entrada 2, com um clipe de padrão Office Binder </li><li> Continue empurrando o PBS 1X até que o fluido alcança a porta de saída do dispositivo de microfluídica. Uma vez que a solução flui para fora da tomada, apertar o tubo de saída com outro clipe de ligante. </li><li> Continue empurrando a seringa de 1 ml até o PBS 1X fluxos de entrada de três, e prenda a porta de entrada 3 com outro clipe Office Binder. O dispositivo microfluídica está agora completamente ferrado. </li></ol><img src="/files/ftp_upload/1656/1656fig1.jpg" alt="Figura 1" /> Figura 1. Esquemática do dispositivo mostrando as entradas para suas respectivas soluções e tomada. 2,3 Preparação de bombas de seringa e solução <ol><li> Para calibrar as bombas siga estes passos: <ol class="lalpha"><li> Para a bomba de seringa grande Harvard (Harvard, PHD Parte 22/2000 # 702000): Define a configuração de diâmetro para 22,5 mm, taxa de fluxo de 600 mL / min. </li><li> Para a bomba de seringa pequena Harvard (Harvard, Pico Mais Part # 702213): Define a configuração de diâmetro para 4,61 mm, vazão 20 l / min. </li></ol></li><li> Preencher uma seringa 30 ml com água deionizada estéril. Isso pode ser feito retirando a solução diretamente do tubo de 50 ml. (Label a seringa) </li><li> Encher uma seringa ml segundo 30 com 2X tampão fosfato-salina (PBS) paraformaldeído a 2% (PFA), utilizando o procedimento descrito no passo 9. (Label a seringa) </li><li> Encha uma seringa com 1 ml de 1X PBS a partir do alíquotas armazenadas na 15 ml tubos cônicos, utilizando o procedimento descrito no passo 9. </li><li> Conecte o 30 ml seringa água deionizada para entrada de 2 e os 30 ml PBS 2X seringa para entrada de 3 (certifique-se de don t armadilha bolhas na tubulação ou outro dispositivo de microfluídica). Retire o grampo de entradas 2 e 3 ea tubulação de saída. Conecte o 1X PBS ml 1 seringa na entrada da 1. Evitar a introdução de bolhas, preenchendo a agulha da seringa com líquido, que liga a seringa, e sacudindo a agulha da seringa para livrar de bolhas. </li></ol> 2,4 lise eritrocitária <ol><li> Conjunto de todas as seringas nas bombas adequada Harvard, conforme descrito abaixo: <ol class="lalpha"><li> Definir as duas seringas 30 ml (uma contendo estéreis, a água de-ionizada e outro com PBS 2X, PFA 2%) juntos na bomba de seringa grande Harvard. </li><li> Coloque a seringa de 1 ml (contendo 1X PBS) na bomba pequena seringa Harvard. </li><li> Coloque a tubulação de saída em um coletor de resíduos (50 ml tubo cônico que é rotulado waste). </li></ol></li><li> Ligue a bomba de seringa grande Harvard (com 30 seringas ml) e deixá-lo correr durante um minuto. (Certifique-se que não há bolhas e nenhum vazamento no aparelho ou na tubulação). </li><li> Ligue a bomba pequena seringa Harvard e deixá-lo correr por mais um minuto. (Certifique-se que não há bolhas e nenhum vazamento no aparelho ou na tubulação). Parar as bombas. O dispositivo microfluídica está agora pronto para ser usado. </li><li> Remova a seringa de 1 ml contendo 1X PBS a partir da pequena bomba de seringa Harvard. </li><li> Depois de obter uma amostra de sangue, preencher a seringa de 1 ml com 0,05 ml de 1X PBS estéril, sem quaisquer bolhas de interceptação. Em seguida, encha a seringa com 0,5 ml de sangue obtidas do tubo Eppendorf. (A seringa de 1 ml conterá agora um volume final de 0,55 ml do sangue e tampão 1X PBS.) Nota: Manter a seringa vertical enquanto enchimento para evitar a mistura de 0,05 ml de PBS com o sangue. </li><li> Conectar a seringa contendo o sangue para uma entrada e montar a seringa com cuidado (não empurrar o sangue através do tubo para dentro do dispositivo) na bomba pequena seringa Harvard (a seringa é montado verticais na bomba). </li><li> Remova o tubo de saída do tubo de descarga e colocar um tubo de centrífuga de 50 ml limpa em seu lugar e defini-lo em um balde de gelo. </li><li> Ligue a bomba de seringa grande Harvard em primeiro lugar e deixá-lo correr por 1 minuto. Começar a recolher a amostra da saída para o tubo de centrifugação de 50 ml. </li><li> Ligue a bomba pequena seringa Harvard. </li><li> Uma vez que a amostra de sangue na seringa de 1 ml foi completamente percorrido através do dispositivo, pare de ambas as bombas. Isso deve demorar aproximadamente 20 minutos. </li><li> Centrifugar a amostra coletada durante 5 minutos a 350 xg à temperatura ambiente com o off freio. </li><li> Remover o sobrenadante, colocando a ponteira no lado oposto do pellet branco. Remover o máximo possível sobrenadante restos celulares, especialmente vermelho, sem perturbar o sedimento branco. </li><li> Ressuspender a amostra em 1 ml de tampão de fluxo (1X PBS, 1% BSA [soro albumina bovina], 2% PFA) pipetando cima e para baixo. Transferência de amostra para um tubo Eppendorf 1,5 ml. </li><li> Microcentrífuga a amostra coletada durante 5 minutos a 4 xg à temperatura ambiente. Use mesma precisão de antes de remover o sobrenadante. </li><li> Ressuspender a amostra em 1 ml de tampão de fluxo em vórtice. </li></ol> 2.5 Preparação para a análise de citometria de fluxo <ol><li> Para análise de amostras por citometria de fluxo, adicione 100 ml de amostra para um tubo de citometria de fluxo. </li><li> Para o 100 mL de amostra adicionar anticorpo específico (garantir a concentração correta). </li><li> Deixar a amostra para incubar por 30 minutos a 40 C. </li><li> Lavar duas vezes com tampão de fluxo antes de citometria de fluxo. Etapa de lavagem inclui a adição de 250 L de tampão de fluxo, vortex, centrifugação por 5 minutos em 350 xg, e remover o sobrenadante com uma volta de fluido do tubo de citometria de fluxo. </li><li> Ressuspender o sedimento em 250 mL de tampão de fluxo. Amostra está pronta para análise por citometria de fluxo. </li></ol> Resultados representante Sangue total foi executada através do dispositivo lise microfluídicos e pós-processamento de protocolo, livrando de eritrócitos circulantes e enriquecendo as células nucleadas (CNCS). Resultados ideais renderá um limpa fluxo gráfico de dispersão com citometria de populações separadas CNC. Exibido abaixo nas figuras 2 e 3 são comparações de dispersão de uma amostra de sangue total sem lise de hemácias e com a lise de eritrócitos pelo protocolo microfluídicos com machados como dispersão para a frente (FSC) versus dispersão lateral (SSC) da luz. Pode ser visto que o protocolo de enriquecimento microfluídicos é necessário obter uma limpeza fluxo gráfico de dispersão citometria. <img src="/files/ftp_upload/1656/1656fig2.jpg" alt="Figura 2" /> Figura 2. Fluxo gráfico de dispersão citometria de amostra de sangue total sem lise de eritrócitos com FSC e SSC eixos. <img src="/files/ftp_upload/1656/1656fig3.jpg" alt="Figura 3" /> Figura 3. Fluxo gráfico de dispersão citometria de circulação de células nucleadas com lise de eritrócitos microfluídicos rotulado por populações com FSC e SSC eixos. Região 1 (vermelho) representa linfócitos, Região 2 (verde) representa monócitos, Região 3 (azul) representa granulócitos e Região 4 (roxo) ainda não foi caracterizado. Remoção de detritos de células vermelhas, com uma pipeta no protocolo também é importante quando for necessário. Abaixo na Figura 4 é um gráfico de dispersão mostrando um excedente de restos de glóbulos vermelhos. Apesar de não nublando o gráfico de dispersão como na Figura 2, deve-se conta para os eventos adicionados devido a restos de hemácias durante a análise de dados. <img src="/files/ftp_upload/1656/1656fig4.jpg" alt="Figura 4" /> Figura 4. Fluxo gráfico de dispersão citometria representando restos de glóbulos vermelhos, vistos como um clusterde eventos na Região 5 (cyan). Células de coloração para os marcadores determinado fenótipo de anticorpos também irá gerar resultados característica. Nas Figuras seguintes são manchas de anticorpos fenótipo por 3 populações de leucócitos distintas: linfócitos, monócitos e granulócitos. Figura 5 representa as populações de linfócitos plotados com machados CD3 e CD4. Monócitos e granulócitos são mostrados nas Figuras 6-7 com eixos como FSC contra CD14 e CD66b, respectivamente. <img src="/files/ftp_upload/1656/1656fig5.jpg" alt="Figura 5" /> Figura 5. Fluxo gráfico de dispersão representando citometria de linfócitos com CD3 e CD4 eixos. <img src="/files/ftp_upload/1656/1656fig6.jpg" alt="Figura 6" /> Figura 6. Fluxo gráfico de dispersão representando citometria de monócitos com FSC e CD14 eixos. <img src="/files/ftp_upload/1656/1656fig7.jpg" alt="Figura 7" /> Figura 7. Fluxo gráfico de dispersão representando citometria de granulócitos com FSC e machados CD66b.

<ol>
	<li>White, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+application+of+microfluidic+leukocyte+enrichment+protocol+in+mild+phenotype+sickle+cell+disease+(SCD.">Clinical application of microfluidic leukocyte enrichment protocol in mild phenotype sickle cell disease (SCD.</a> Biomedical Microdevices. 11 (2), 477-483 (2009).</li><li>Sethu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+Flow+Microfluidic+Device+for+Rapid+Erythrocyte+Lysis.">Continuous Flow Microfluidic Device for Rapid Erythrocyte Lysis.</a> Anal. Chem. 76 (21), 6247-6253 (2004).</li></ol>

Um protocolo de lise automatizado microfluídicos de sangue tem sido relatado. No âmbito do protocolo são passos críticos dignos de nota. Na seção P.2.1, é importante para descartar o frasco de sangue coletadas primeira vez que a amostra contém células endoteliais desalojado madura agindo como falsos positivos. Certifique-se também para executar o amostra de sangue dentro de uma hora da coleta. Priming o dispositivo microfluídicos na seção p.2.2, é importante inicialmente livrar das bolhas. Além disso, dev...

automated microfluidic blood lysis protocol for enrichment of circulating nucleated cells

Neste relatório, o protocolo para um dispositivo de lise microfluídicos sangue automatizado é detalhado. Circulantes de células nucleadas (CNCs), incluindo leucócitos e células endoteliais, fornecem a plataforma ideal para um status atualizado sobre o estado imunológico de um indivíduo. O protocolo permite microfluídicos para o enriquecimento de CNCs sem perda seletiva de células e preparação de amostras a variabilidade devido ao usuário mediada por etapas. Resumidamente, o protocolo inclui a fabricação de dispositivos, coleta de amostras, a configuração de dispositivos e executando o sangue através da câmara microfluídicos. Dentro do dispositivo de sangue total é rapidamente misturada com água deionizada por cerca de 10 segundos em um x 50 150 micron micron canais microfluídicos. Neste tempo eritrócitos são lisados ​​período devido às condições hipotônica. Estruturas de espinha de peixe na parte inferior do canal de garantir a mistura completa e exposição de células a um ambiente constante. Células restantes são devolvidos às condições isotônicas na saída do dispositivo, fixa usando paraformaldeído 2%, centrifugado para separar fragmentos de eritrócitos de CNCs, e suspenso em tampão de fluxo para a coloração e análise por citometria de fluxo. Os resultados mostram limpa fluxo parcelas citometria de dispersão com populações CNC salvo. Importância deste dispositivo e protocolo vem no estudo e compreensão da patogênese da doença através da análise de populações CNC. Por isso, a automação, a eficácia ea simplicidade do protocolo microfluídicos são demonstradas.

Watch this Scientific Journal Video about Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells at JoVE.com

Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

Um dispositivo automático microfluídicos foi desenvolvido para a circulação de enriquecimento de células nucleadas do sangue periférico, através de lise de eritrócitos que garante o isolamento de amostras de alta qualidade sem perda de células.

Protocolo de Lise automatizado microfluídicos de sangue para Enriquecimento de células nucleadas circulantes

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and ...

automated-microfluidic-blood-lysis-protocol-for-enrichment

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JoVE Journal

Biology

12.3K visualizações.  University of Louisville. Um dispositivo automático microfluídicos foi desenvolvido para a circulação de enriquecimento de células nucleadas do sangue periférico, através de lise de eritrócitos que garante o isolamento de amostras de alta qualidade sem perda de células.

Vídeo: Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

Enriquecimento das células NK do sangue humano com o Kit RosetteSep de StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

Biologia

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Microfluídicos Co-cultura de células epiteliais e bactérias para a Investigação Solúvel sinal mediada por interações

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

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 ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

Proteínas Análise de imunofluorescência de endógenos e exógenos Centromere-cinetócoro

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

Usando dispositivos microfluídicos medir Longevidade e Celular fenótipos em células individuais levedura de brotamento

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Uma interface de usuário gráfica para rastreamento assistido por software da concentração de proteínas em protrusões celulares dinâmicas

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

Um protocolo para o uso de Gene definir análise de enriquecimento para identificar o modelo Animal adequado para investigação de translação

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the lymph node microenvironment in B-cell non-Hodgkin&#39;s lymphoma is analyzed. Subsets of circulating T-follicular helper cells (cTfh), the circulating memory compartment of Tfh cells in the blood, are also perturbed in disease and therefore represent potential novel predictive biomarkers. Peripheral blood-based testing is advantageous because it is relatively non-invasive and allows simple serial monitoring.This article describes a method for isolating CD4+ T-cells from human blood, and further analysis by flow-cytometry to enumerate cTfh cells and the proportions of their various subsets (cTfhPD-1-/+/hi, cTfh1,2,17 and cTfh1/17). The level of these subsets was then compared between normal subjects and patients with lymphoma. We found that the method was robust enough to obtain reliable results from routinely collected patient material. The technique we describe for the analysis can be easily adapted to cell sorting and downstream applications such as RT-PCR.

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Here, a protocol for the isolation and characterization of CD4+ T-cell subsets from human peripheral blood is described. Purified CD4+ T-cells are analyzed by flow cytometry to determine proportions of T-follicular helper cell subsets.

Isolamento de CD4+ T-células e análise de circulação folicular-T Helper (cTfh) célula subconjuntos de citometria de fluxo de 6 cores usando o sangue periférico

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Isolamento e enriquecimento de células progenitoras epiteliais pulmonares humanas para a cultura organoide

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Quantification of Circulating Pig-Specific DNA in the Blood of a Xenotransplantation Model

Xenotransplantation is a feasible method to treat organ failure. However, how to effectively monitor the immune rejection of xenotransplantation is a problem for physicians and researchers. This manuscript describes a simple and effective method to monitor immune rejection in pig-to-mouse cell transplantation models and pig-to-monkey artery patch transplantation models. Circulating DNA is a potentially non-invasive biomarker for organ damage. In this study, circulating pig-specific DNA (cpsDNA) was monitored during xenograft rejection by quantitative real-time PCR (qPCR). In this protocol, porcine specific primers were designed, plasmids-containing porcine specific DNA fragments were constructed, and standard curves for quantitation were established. Species-specific primers were then used to quantify cpsDNA by qPCR in pig-to-mouse cell transplantation models and pig-to-monkey artery patch transplantation models. The value of this method suggests that it can be used as a simple, convenient, low cost, and less invasive method to monitor the immune rejection of xenotransplantation.

In this protocol, porcine specific primers were designed, plasmids-containing porcine specific DNA fragments were constructed, and standard curves for quantitation were established. Using species-specific primers, cpsDNA was quantified by qPCR in pig-to-mouse cell transplantation models and pig-to-monkey artery patch transplantation models.

Quantificação do DNA específico do porco circulante no sangue de um modelo de xenotransplante

Xenotransplantation is a feasible method to treat organ failure. However, how to effectively monitor the immune rejection of xenotransplantation is a ...