Este trabalho foi financiado pelo National Institutes of Health Grant R01 AI088209 (para KAH). Agradecemos também a Universidade de Minnesota Citometria de Fluxo de recursos.

Neste estudo, os adultos - foram usados ​​(6 8 semanas) do sexo feminino C57BL / 6. Ratos foram adquiridos no Instituto Nacional do Câncer e mantidos sob condições específicas livres de patógenos. A Universidade do Comitê de Cuidados e Uso de Animais Institucional Minnesota (IACUC) aprovou toda a experimentação animal. 1 Preparação de Instrumentos e amortecedores <ol><li> Preparar a solução de enzima (meio RPMI 1640 com 0.05% [w / v] de Liberase TH e 100 U / ml de DNase I), tampão rico em albumina (1X PBS, [Ca 2 + / Mg 2 + livre], com 0,5% de soro de bovino albumina e 2 mM de ácido etilenodiamino tetra-acético [EDTA]), de tampão de FACS (PBS 1X [Ca 2 + / Mg 2 + livre] contendo soro de bovino fetal a 1% [FBS] e 0,02% de NaN 3), separação por FACS tampão (PBS 1X [Ca 2 + / Mg 2 + livre] contendo 2% de FBS), e meio RP10 (RPMI-1640 fornecido com 10% de FBS). </li><li> Prepare 10 placas panning. Preparar a solução de revestimento de 40 ml (0,01 mg / ml de Laveia anti-IgG de rato em 0,1 M de tampão de NaHCO3), e adicionar 4 ml de solução de revestimento em cada 100 x 15 mm placa, redemoinho e incubar a 4 ° CO / N. Escorrer solução de anticorpo e lavar a placa com 5 ml de PBS 1x por 2 vezes, agite vigorosamente entre lavagens. Armazenar as placas preparadas a 4 ° C. </li></ol> 2 Colheita de Thymus Tissue <ol><li> Eutanásia rato numa câmara de CO 2. Coloque o animal em suas costas em uma esteira dissecação, fixar os pés no tapete e molhar a pele por aspersão com etanol 70%. </li><li> Fazer uma incisão na linha média através da pele, a partir de cima da abertura da uretra para cima para o maxilar inferior com uma tesoura e alargar a incisão para baixo para os joelhos em ambos os lados. A incisão se parece com uma cabeça para baixo &quot;Y&quot;. Retire a pele solta de volta nas laterais, e fixá-lo para o tapete dissecção. </li><li> Perfurar o diafragma do xifóide, cortar as costelas de cada lado até cerca da clavícula, em seguida, levante os reforços comfórceps e fixá-lo para o tapete dissecção. O timo é apenas abaixo das costelas, e se parece com dois lobos brancos finos que recobrem o coração (Figura 1). Tecido conjuntivo Disconnect torno do timo, retire com cuidado e retire a par de lobos do timo com uma pinça serrilhada curvas. Lóbulos timo lugar em uma placa de 6 poços contendo 5 ml de meio RPMI-1640. </li></ol> 3 Preparação de Células do estroma tímico <ol><li> Aparar qualquer tecido adiposo e conjuntivo circundante e transferir os lóbulos timo para um novo poço da placa de 6 poços contendo 5 ml de solução de enzima recentemente preparar. Fazer pequenas incisões nos lóbulos do timo com uma tesoura fina, e incubar a placa a 37 ° C durante 20 min. </li><li> Pipeta suavemente digerir o tecido do timo e para baixo 2-3 vezes usando 5 ml de pipeta para dissociação de tecidos. Recolher a fracção de sobrenadante num tubo de 50 ml contendo 10 ml de tampão frio rico em albumina para neutralizar as enzimas. Mantenha o tubo de coleta no gelo. Adicionar 2,5solução de enzima ml para o tecido restante e incubar a placa a 37 ° C durante 15 min. </li><li> Realizar a agitação mecânica suave com uma seringa de 3 ml e agulha de 18 G para quebrar agregados. Piscina sobrenadante para o tubo de coleta. Adicionar solução de 2,5 ml de enzima no tecido remanescente e incubar a placa a 37 ° C durante 15 min. </li><li> Repetir a agitação mecânica suave com uma seringa de 3 ml e uma agulha G 25, e incuba-se a placa adicional 5 - 10 min. </li><li> Após a digestão completa, piscina do sobrenadante para o tubo de coleta. Centrifugar o tubo de recolha a 400 xg, 4 ° C durante 8 minutos para recolher as células. Aspirar o líquido e suspender as células peletizadas em 10 ml de tampão ricos em albumina e filtrar a amostra através de malha de 100 milímetros. Contagem de células utilizando um hemocitómetro. Em seguida, realizar a análise de citometria de fluxo (Seção 4) ou TEC enriquecimento (Seção 5). </li></ol> 4 Identificação de TEC por Citometria de Fluxo </p&gt; <ol><li> Preparar células para uma concentração final de 4 x 10 6 culas por 100 ul em tampão de SCAF para a coloração de uma placa de ensaio de 96 poços de fundo redondo e manter a placa em gelo. Adicionar 20 ul solução de bloqueio de Fc (CD16 anti-anticorpo / CD32 em 10 ug / ml em tampão de SCAF) para as células e incuba-se as células durante 10 minutos em gelo. Girar a placa a 400 x g, 4 ° C durante 3 min. Descarte o líquido das cavidades sacudindo o rosto placa para baixo em uma pia. </li><li> Suspender as células peletizadas em 100 mL de cocktail de anticorpos (anti-CD45, -EpCAM, classe II -MHC, e anticorpos -Ly51, e a lectina UEA-1 marcados com diferentes fluorocromos no fabricante ou recomendadas testadas concentrações óptimas de cada anticorpo em tampão FACS ), e incuba-se as células em gelo durante 20 min no escuro. Para compensação, manchar um x 10 6 células com cada fluorocromo individual. </li><li> Adicionar 100 ul de tampão de SCAF a cada poço e a placa de girar a 400 xg, 4 ° C durante 3 min. Repetir o passo de lavagemtempo, e em seguida ressuspender as células em 100 ul de tampão de FACS. Transferência das células para um tubo de FACS de fundo redondo de 12 x 15 mm, encheu-se com 300 ul de tampão de FACS. Adquirir amostras no citômetro de fluxo e análise de dados usando o software de análise FACS. Estratégia gating TCE é mostrado na Figura 2. </li></ol> 5. Enriquecimento da TEC garimpando <ol><li> Girar as células do timo (preparados na Seção 3) a 400 xg, 4 ° C por 5 min. Aspirar o líquido e suspender as células peletizadas em tampão FACS triagem a uma concentração de 2 x 10 8 culas por ml em 15 ml ou um tubo de 50 ml. Adicionar anticorpos anti-CD90.2 (Clone 30-H12) (ou anti-CD45), a uma concentração final de 2,5 ug / ml de suspensão de células e incubar as células suavemente num misturador rotativo a 4 ° C durante 30 min. A seguir à incubação, lava-se com uma 5 - volume de meio RP10 10x e centrifugar o tubo a 400 xg, 4 ° C durante 5 min. </li><li> Aspirar o líquido e voltar a suspender as células aglomeradas emMeio RP10 a uma concentração de 1 x 10 7 por ml. Adicionar 5 ml de suspensão de células para cada placa revestida panning (preparada no Passo 1.3), redemoinho e incubar durante 30 min à temperatura ambiente. Agite vigorosamente; transferir o sobrenadante para um tubo cónico. Lavar a placa duas vezes com o meio RP10 e reunir os sobrenadantes dentro do tubo de coleta. </li><li> Centrifuga-se o tubo de recolha a 400 xg, 4 ° C durante 5 min. Aspirar o líquido e voltar a suspender as células aglomeradas em cinco ml de meio RP10. Adicionar a suspensão de células para uma nova placa de deslizamento, agite e incube durante 30 min à temperatura ambiente. Coletar as células lavar o prato e reuni-los em um tubo cônico. Uma vez que são enriquecidas TEC, rodar para baixo as células a 400 xg, 4 ° C durante 5 min e suspender as células em 5 ml de tampão de FACS triagem. </li></ol> 6. Purificação da TEC por separação de células <ol><li> Contagem de células utilizando um hemocitómetro e preparar a suspensão de células a uma concentração de 4 x 10 7 células em tampão de SCAF triagem. Adicionar anticorpos described na Secção 4.4 em solução de células e incubar as células suavemente num misturador rotativo a 4 ° C durante 30 min. Após a coloração, lavar e suspender as células a uma concentração de 10 x 10 6 culas por mL de tampão de FACS triagem. </li><li> Ordenar subconjuntos TEC através de um bocal de 100 pM utilizando separação de células activadas por fluorescência. Célula classificados são recolhidos em 30% (v / v) de FBS em meio RPMI-1640). Uma vez que separação de células é feito, centrifugar células a 400 xg, 4 ° C por 5 min e se preparar para análise a jusante. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

No protocolo, os passos críticos são a preparação de células do estroma do timo (ponto 3) e o enriquecimento da TEC (Seção 4). É altamente recomendável que a solução enzimática fresca é preparada de cada vez e que o tecido é tratado o mais rapidamente possível. Para timos reunidas, optimizar o volume da solução de enzimas é necessária, dependendo do número de timos. Se qualquer resíduo de tecido é deixado após o processamento, a adição de mais solução enzimática e extensão do tempo de incuba...

No início do desenvolvimento das células T, de medula óssea hematopoiéticas derivadas de células progenitoras pluripotentes são recrutados para o córtex do timo, submetido a compromisso de linhagem T e tornar-se precursores de células T 1. No córtex, precursoras de células T CD4 e CD8 duplo negativos (DN) timócitos expandir e diferenciar em imaturo CD4 e CD8 duplo positivo (DP) timócitos, formando uma grande piscina de progenitores com receptores de células T altamente variável 1. Apenas um seleto subconjunto MHC-restrito de células DP vai se tornar único positivos (SP) timócitos CD4 ou CD8, migram para a medula do timo, e se diferenciar em células T maduras funcionalmente competentes, um evento que é chamado de seleção como positiva 2 6. Em contraste, os clones de timócitos auto-reactivos sofrem de selecção negativa e são removidos por meio de apoptose, convertidas em células T reguladoras para a auto-tolerância, ou desviado para linfócitos intra-epiteliais para fins que ainda não são claras 3,7-10. No timo, as células do estroma de timo formar um microambiente único fornecimento de sinais para esses vários destinos de desenvolvimento de células T 5,11,12. As células do estroma de timo são compostas por células epiteliais tímicas (TEC) - incluindo TEC corticais (CTECs TEC) e medulares (mTECs), células dendríticas, macrófagos, fibroblastos, células endoteliais, pericitos derivados da crista neural e outras células mesenquimais 13-15. Entre estes, TEC são cruciais nas várias fases de desenvolvimento das células T 1,2,16,17. No entanto, a falta de uma forma robusta para isolar TEC tem dificultado uma compreensão abrangente de suas funções 16. Em particular, CTECs, que formam uma rede tridimensional circundante progenitores no córtex, são essenciais para a seleção positiva 13,18,19 por razões que ainda não estão claras. Estudos anteriores forneceram pistas sobre a heterogeneidade eo papel da TEC, principalmente contando com ferramentas morfológicas e histológicas 13 </sup&gt;. Recentemente, os papéis únicos de subconjuntos TEC foram abordados por abordagens genéticas em modelos de ratos 12,20. A forma robusta e reproduzível para isolar TEC é fundamental para alcançar a caracterização imparcial de subconjuntos do TCE, avaliação quantitativa e qualitativa das funções do TCE, e esclarecimento de mecanismos como CTECs apoiar seleção positiva. Devido à raridade da TEC no timo e as interações apertadas formam no órgão intacto, o isolamento da TEC tem sido um desafio. O protocolo aqui descrito é baseado em descobertas anteriores, atualmente reagentes disponíveis, técnicas e conhecimento da estrutura do timo e composição estromal. Há quase duas décadas, vários procedimentos foram relatados desagregar tecidos timo 21-27, em que diferentes enzimas foram utilizadas durante a digestão, incluindo tripsina, colagenase e Dispase. Gray et al. comparação dessas enzimas em sua precedure 28, e relataram uma melhoria da method com a digestão de várias etapas de coquetéis enzimáticos 29 que se tornaram amplamente utilizados 20,30. No entanto, este método envolve um tempo de preparação longos e complexos processos de digestão e resulta em última variável o número de células e as proporções de TEC ainda na mesma coorte de murino 29,30. Vários anos atrás, Liberase enzima grau de investigação, contendo altamente purificado colagenase e protease neutra começou a ser utilizado na dissociação tecido do timo 30. Aqui, nós descrevemos um protocolo baseado em digestão Liberase, com processos de separação mecânica optimizadas, que produz um elevado número de TEC viáveis ​​a partir de tecidos do timo do rato. . Para enriquecer células estromais dissociados, estudos anteriores têm usado gradientes de densidade ou separação magnética talão 21,29. No entanto, ambos os métodos de causar grave perda de determinadas populações de células do estroma de timo, especialmente a população de CTECs 29. Eliminação citotóxica e técnicas de panning <sse&gt; 31,32 têm sido amplamente utilizadas para a depleção ou separação de linfócitos no campo imunológica 12,31. Após a comparação destas técnicas, foi estabelecido o protocolo de panning atual para o enriquecimento da TEC. A condição suave durante o processo de enriquecimento leva a menos morte celular e imparcial e uma maior recuperação do TCE. A suspensão de células do timo isolado descrito na Seção 3 pode ser aplicado diretamente para análise por citometria de fluxo para a identificação e caracterização dos subconjuntos TEC e células dendríticas. A Seção 4 descreve uma forma simples e útil para identificar subconjuntos TEC utilizando fluxo multiparâmetro citômetro. Para experiências que procuram obter CTECs purificadas ou mTECs, TEC enriquecimento e células de triagem procedimentos podem ser encontradas na Seção 5 e 6.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:487px;">Anti-mouse EpCAM</td>
			<td style="width:153px;">eBioscience</td>
			<td style="width:133px;">XX-5791-YY*</td>
			<td style="width:163px;">Clone G8.8</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-mouse MHC II</td>
			<td>eBioscience</td>
			<td>XX-5321-YY*</td>
			<td>Clone M5/114.15.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-mouse Ly51</td>
			<td>eBioscience</td>
			<td>XX-5891-YY*</td>
			<td>Clone 6C3</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UEA-1</td>
			<td>Vector Laboratory</td>
			<td>FL-1061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cytometer</td>
			<td>BD Biosciences&#160;</td>
			<td>LSRFortessa</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cell sorter</td>
			<td>BD Biosciences&#160;</td>
			<td>FACSAria</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FACS tubes</td>
			<td>BD Biosciences</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cytometry analysis software</td>
			<td>TreeStar - Flowjo</td>
			<td>FlowJo v7/9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">*XX varies by fluorochrome and YY varies by vial size.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, identification, and purification of murine thymic epithelial cells

O timo é um órgão vital para o desenvolvimento de linfócitos T. De células do estroma do timo, as células epiteliais do timo (TEC) são particularmente crucial em vários estágios de desenvolvimento de células T: compromisso de células T, de seleção positiva e negativa de seleção. No entanto, a função de TEC no timo permanece incompletamente compreendida. No artigo, nós fornecemos um método para isolar subconjuntos TEC de timo fresco rato usando uma combinação de ruptura mecânica e digestão enzimática. O método permite que as células do estroma de timo e de timócitos de ser eficientemente libertado a partir de células de células e ligações célula-matriz extracelular e para formar uma suspensão de célula única. Utilizando as células isoladas, multiparamétrica de citometria de fluxo pode ser aplicado para a identificação e caracterização de TEC e células dendríticas. Porque TEC são uma população rara de células no timo, que também descrevem uma forma eficaz para enriquecer e purificar a TEC, esgotando os timócitos, o tipo de célula mais abundante no timo. Folloala o enriquecimento, a selecção de células de tempo pode ser reduzida de forma que a perda de viabilidade das células pode ser minimizada durante a purificação de TEC. As células purificadas são adequados para diversas análises a jusante como o Real Time-PCR, Western blot e perfil de expressão gênica. O protocolo irá promover a investigação da função do TCE e, assim como o desenvolvimento de reconstituição in vitro de células T.

Usando este protocolo, um órgão timo foi removido a partir de um rato adulto (Secção 2) e uma suspensão de células do timo, foi preparado como descrito no ponto 3 a suspensão de células obtida consistiu de timócitos, células de estroma derivadas de hematopoiéticas e células estromais não hematopoiéticas. CD45 é um marcador de pan-hematopoiético expressos em ambos os timócitos e células do estroma hematopoiético, tais como macrófagos e células dendríticas. Em contraste, os TEC não são de origem he...

Watch this Scientific Journal Video about Isolation, Identification, and Purification of Murine Thymic Epithelial Cells at JoVE.com

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Descrevemos aqui um método eficaz para o isolamento, identificação e purificação de células epiteliais do timo do rato (TEC). O protocolo pode ser utilizada para estudos da função do timo para o desenvolvimento normal das células T, disfunção do timo, e a reconstituição de células T.

Isolamento, identificação e purificação de células epiteliais do timo murino

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

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Research

JoVE Journal

Immunology and Infection

31.7K visualizações.  University of Minnesota.  Descrevemos aqui um método eficaz para o isolamento, identificação e purificação de células epiteliais do timo do rato (TEC). O protocolo pode ser utilizada para estudos da função do timo para o desenvolvimento normal das células T, disfunção do timo, e a reconstituição de células T. 

Vídeo: Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

Aplicação de um rato ligado patch Peyer Assay alça intestinal para avaliar absorção pelas células bacterianas M

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Imunologia e Infecção

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Isolamento de citometria de fluxo de células primárias Murino Tipo II epiteliais alveolares de Estudos funcionais e Molecular

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

Isolamento, processamento e análise de células gengivais murino

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

O isolamento de células dendríticas mielóides e células epiteliais de timo humano

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Isolamento de Murino Linfonodo células estromais

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

Grande Escala da Purificação de suínos ou bovinos fotorreceptores Outer Segmentos para a fagocitose Ensaios sobre Retinal Pigment células epiteliais

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

Isolamento e Transplante de diferentes envelhecidas murino tímicos enxertos.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

Promover 3-D agregação de células FACS purificada epiteliais tímicos com EAK 16-II / EAKIIH6 de auto-montagem Hidrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

Purificação de DNA Viral para a identificação de proteínas Viral e celulares associadas

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.