The authors thank Sanjiv Luther and colleagues for helpful discussions in establishing the current lymph node digestion protocol. This work was supported by SNF grants PPOOA-_119204 and PPOOP3_144918 to S.W.R.

Nesta publicação de vídeo e protocolo, todos os procedimentos com animais foram realizados de acordo com o protocolo de animais aprovados pela Autoridade Cantonal Basel-Stadt, Suíça. 1. Linfonodos Preparação e Digestão <ol><li> Pré-aquecimento de água num copo a 37 ° C num agitador magnético com placa de aquecimento. </li><li> Prepare médio básico como se segue: DMEM (sem piruvato) suplementado com 2% de FCS, 1,2 mM CaCl2 e Pen / Strep (100 unidades de penicilina, 100 ug de estreptomicina). </li><li> Esterilizar todos os instrumentos de dissecção antes do uso. </li><li> Eutanásia camundongos linfonodo doadores por asfixia com CO2 e assepticamente dissecar os gânglios linfáticos. Não dissecar a gordura ao redor. NOTA: Este protocolo é otimizado para o nó periférico de drenagem linfática da pele (inguinal, braquial, axilar). </li><li> Coloque gânglios linfáticos em uma placa de Petri esterilizada, contendo 2 ml de meio de base de gelo frio. </li><li> Interromper o linfonodo capsule usando duas agulhas 25 G fixos em 1 ml seringa. </li><li> Transferir o tecido de linfonodos interrompido num tubo de polipropileno de 5 ml de fundo redondo contendo 750 ul meio básico complementado com 1 mg / ml de colagenase IV, e 40 ug / ml de ADNase I. </li><li> Adicionar um agitador magnético estéril em cada tubo. </li><li> Colocar o tubo no recipiente com 37 ° C pré-aquecido de água e agita-se os tubos a uma velocidade lenta (1 volta / seg) durante 30 min. </li><li> Remover o tubo do agitador magnético com placa de aquecimento e deixar fragmentos de linfonodos resolver. </li><li> Remova cuidadosamente o sobrenadante enriquecido em &quot;células não-estromal&quot;. NOTA: Se a análise de T, B, células dendríticas e CD45 - gp38 - CD31 - está prevista, salvar a fração &quot;células não-estromais&quot;. </li><li> Lavar o tecido restante linfonodo uma vez com 750 ul meio básico. NOTA: Esta etapa é necessária somente se a trabalhar com camundongos imunocompetentes. </li><li> Vamos fragmentos de linfonodos resolver. </li><li> Remover ofracção flutuante de células não-estromal. </li><li> Adicionar aos fragmentos de nódulos linfáticos meio básico de 750 ul suplementadas com 3,5 mg / ml de colagenase D e 40 ug / ml de ADNase I. </li><li> Colocar o tubo de volta na taça que contém a 37 ° C pré-aquecido a água. </li><li> Digerir o tecido do linfonodo para 5 min, enquanto lentamente mexendo. </li><li> Linfáticos fragmentos de tecido nó desagregados por pipetagem e misturando 700 mL para 10 ciclos em máxima velocidade usando uma pipeta multicanal automatizada. NOTA: Isso atrapalha linfonodo para melhorar a digestão. </li><li> Colocar o tubo de volta no recipiente com água a 37 ° C pré-aquecido. </li><li> Fragmentos de tecidos de linfonodo Digest para mais 10 min, enquanto lentamente agitação. </li><li> Desagregar fragmentos de tecidos de nódulos linfáticos por pipetagem e mistura por 99 ciclos a velocidade máxima utilizando uma pipeta multicanal automatizada. </li><li> Adicionar 7,5 mL de 0,5 M de EDTA para garantir a manutenção da suspensão de célula única. </li><li> Fragmentos de tecidos de linfonodo desagregados por tubotting e mistura por 99 ciclos a velocidade máxima utilizando uma pipeta multicanal automatizada. </li><li> Adicionar meio básico de 750 ul e as células passam através de uma malha de nylon de 70 ^ m. </li><li> Centrifugar a suspensão de células de 5 min a 1500 xg, 4 ° C. </li><li> As células do estroma pode ser agora usado para posterior análise. </li></ol> 2 Coloração de Linfonodo células estromais <ol><li> Use uma combinação de anti-CD45, anti-podoplanina (gp38), os anticorpos anti-CD31 para reconhecer com sucesso células TRC, LEC, BEC e DN. </li><li> Incubar o tecido linfático nó digerido com vivo inoperante rastreador /, anti-CD45-FITC (1: 200), anti-gp38-PE (1: 200), anti-CD31-APC (1: 200) em 100 uL de HBSS contendo 2 % FCS para, pelo menos, 20 min, 4 ° C, no escuro. </li><li> Lave as células adicionando 500 l de HBSS contendo 2% de FCS </li><li> Centrifugar a suspensão de células a 3 minutos a 1500 xg, 4 ° C. </li><li> Re-suspender em 100 uL de HBSS contendo 2% de FCS. </li><li> Executar as células marcadas num citómetro de fluxo equed, com as seguintes óptica: fonte de excitação com até três lasers: azul (488 nm, arrefecido a ar, de 20 mW de estado sólido), vermelho (633 nm, 17 mW HeNe), e violeta (405 nm, 30 mW de estado sólido) . </li><li> Portão de CD45 - células para excluir as células hematopoiéticas. </li><li> Singlets Portão (FSC-W) e de células vivas (VIVO / rastreador DEAD) para excluir doublets e células mortas. </li><li> Plot gp38 contra CD31 para visualizar TRC (gp38 + CD31 -), LEC (gp38 + CD31 +), BEC (gp38 - CD31 +) células (ver Figura 1) e DN (gp38 - - CD31). </li></ol>

<ol>
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The authors declare they have no competing financial interests.

O estudo das células do estroma dos nódulos linfáticos recentemente tornou-se um foco de investigação devido ao desenvolvimento de dois protocolos de digestão publicados 6,13. Ambos os protocolos são adequados para obter único nó de linfa de células estromais, mas diferem na utilização de enzimas de digestão e tempo de digestão. Uma vez que as células estromais e seus marcadores de superfície são sensíveis à digestão enzimática e tensão mecânica, um protocolo optimizado é necessária. O isolamento das células do nódulo linfático do estroma viáveis ​​de linfonodo recentemente dissecados é o primeiro passo para executar fenotípica e análise funcional. Portanto, é importante para digerir cuidadosamente com a concentração definida de enzimas, a uma temperatura estável e durante o tempo indicado. Dois protocolos de digestão foram descritos 6,13, no entanto, com os passos de digestão relativamente longos. Para reduzir o tempo de digestão, a desagregação mecânica foi incluida e padronizados utilizando uma pipeta multicanal. Em<html> e das vantagens do protocolo actual é que ele permite o isolamento de células do estroma, em apenas 45 minutos, minimizando assim o seu tempo de incubação com as enzimas de digestão. Além disso, sob agitação constante durante a digestão permite o acesso óptimo das enzimas de digestão para a estrutura de nó de linfa. Além disso, o volume foi reduzido a digestão de 750 ul minimizando a quantidade de enzima utilizada. Força mecânica excessiva perturba junções apertadas, mas pode causar a morte de células ao mesmo tempo. Por esta razão, nós testamos várias combinações de ciclos de re-suspensão e diferentes dispositivos de dissociação. Nós descobrimos que a utilização de uma pipeta de canais múltiplos automatizado permite a dissociação em 2 aplicações de 99 ciclos; aplicar uma pressão constante para as células e isto resultará em suspensão de células individuais, com a viabilidade óptima. Quatro a seis amostras pode ser misturada e pipetada, ao mesmo tempo, reduzir o tempo do nó de linfa de isolamento de células do estroma. EOR &quot;&gt; O protocolo de digestão tem de manter a expressão na superfície de quase todas as moléculas;. somente desta maneira concomitante com a coloração de vários marcadores podem revelar se uma população homogénea ou heterogénea, por exemplo, o uso de BP-3 e CD35 na TRC gp38 + CD31 -. permite a visualização e isolamento de medular TRC (Sanjiv Luther, comunicação pessoal) Além disso, a fracção de DN mal caracterizados foi ainda caracterizado como contendo células Aire + e pericitos, como células positivas para a integrina α7 (revisto em 7, 13). Portanto, o presente protocolo foi comparado aos publicados. Conforme mostrado nos resultados, frequências e de números de células estromais de linfonodos semelhantes foram obtidos com o protocolo atual em comparação com os publicados. Curiosamente, a frequência eo número de isolados TRC Foram mais alto usando o protocolo Fletcher sugerindo uma melhor dissociação das estruturas TRC que no protocolo atual eProtocolo Link. Além disso, a expressão de várias moléculas de superfície foi reforçada com o protocolo atual em relação ao protocolo de Fletcher. Estas diferenças no método de digestão pode ser a razão para um pouco diferentes padrões de expressão nos estudos com células do estroma dos nódulos linfáticos 6,19,21. Pode ser útil para testar diferentes protocolos de digestão, por causa das vantagens de cada protocolo. O protocolo de corrente pode ser melhor para a análise da expressão de superfície, enquanto que o protocolo Fletcher pode ser útil para o isolamento de um elevado número de células viáveis ​​para análises de transcrição (tal como publicado em Malhotra et al. 7). Portanto comparando os três protocolos de digestão pode ser importante para obter os melhores resultados após o isolamento de células do estroma dos nódulos linfáticos e devem ser vistas como complementares.

Os linfonodos são compartimentos especializados onde as respostas imunes adaptativas contra-antígenos próprios estrangeiros e são iniciadas e coordenadas. O processo aqui apresentado descreve uma digestão enzimática curto combinado com pipetagem automatizada mecânica para obter linfonodo suspensão de células individuais e obter acesso às células viáveis ​​do estroma de nódulos linfáticos, que mantêm a expressão de várias moléculas da superfície. Formar células do estroma linfonodo cadafalso do linfonodo e cumprir três funções principais: primeiro eles filtram líquidos do corpo para provar antígenos, patógenos e sua patógeno padrão molecular associado (PAMPs), bem como as citocinas e perigo associado padrão molecular (amortece) presente no corpo. Em segundo lugar, eles atraem e instruir as células apresentadoras de antígenos (APC) e linfócitos para interagir e iniciar respostas imunes adaptativas; e em terceiro lugar, que proporcionam um ambiente estrutural para a homeostase e diferenciação dos linfócitos 1-3. Durante a inflamação células do linfonodo estromais produzem fatores de crescimento, citocinas e quimiocinas, adaptar-se, assim, o inchaço organizar a interação entre células dendríticas (DCs), T e células B. A orquestração das respostas imunitárias só é possível devido a arquitectura estrutural complexo formado por diferentes populações de células do estroma. As células do estroma de nódulos linfáticos são células CD45 negativas e pode ser distinguida da expressão de CD31 ou gp38 em células fibroblásticas e endoteliais 1 -6. GP38 + CD31 - define as células da zona T reticulares (TRC, também conhecidos como FRC: células fibroblásticas reticulares), gp38 + CD31 + define células endoteliais linfáticas (LEC), gp38 - CD31 + define as células endoteliais do sangue (BEC). Além disso, a caracterização das subpopulações revelou a existência de outras células do estroma de nódulos linfáticos. De facto, uma pequena população de células-pericyte como foi caracterizado nagp38 - CD31 - população 7. Portanto, a adaptação do procedimento de isolamento é vantajosa para a identificação e caracterização das propriedades funcionais diferentes de células do estroma de nódulos linfáticos. Antes do desenvolvimento de linfonodo células estromais digestão protocolos que o estudo de células estromais de linfonodos foi limitado a observações in situ usando secção de tecido e microscopia. No entanto, estudos estruturais e funcionais apresentaram características importantes de células estromais de linfonodos. As células do estroma de nódulos linfáticos são associados com podoplanina, colagénio e proteínas (ECM) de matriz extracelular de modo a formar uma estrutura tridimensional complexa 3 chamado sistema de conduta, a qual transporta as proteínas linfáticas e massa molecular associada menor do seio subcapsular do nó de linfa para o alto endotélio vênulas na zona de células T 8. DCs estão em contato com células do estroma e pode ser observado saliente no con tubularestrutura duit a amostra de fluido e detectar antigénios 8. A interação das células do estroma dos nódulos linfáticos (TRCs e LECs) com DCs é mediada pela liberação e apresentação das quimiocinas CCL21 e CCL19 9,10. CCL19 e CCL21 são reconhecidos pelo receptor CCR7 facilitar DCs e as células T migrem para a zona das células T linfonodo 4,11. Apesar de usar quimiocinas semelhantes, as células T e DCs têm diferentes rotas de migração para os linfonodos 12. Mais tarde, utilizando a digestão enzimática do nódulo linfático e isolamento de células do estroma de nódulos linfáticos puros, estudos funcionais foram realizados sobre o papel dos diferentes células do linfonodo estroma e a sua capacidade para interagir com DC e células T / B 6,13. Em primeiro lugar, a interferência entre as células produtoras de IFN-T efectoras e células estromais γ nódulos linfáticos induz a produção do óxido nítrico metabolito mostrado para amortecer as respostas das células T e a proliferação dos órgãos linfóides secundários 14-16. Em segundo lugar, n linfaAs células do estroma ode ter sido relatado para suportar a diferenciação de subconjuntos de DC de regulação através da produção de IL-10, 17, e para modular a homeostase da célula T ingénuos através da produção de IL-7 6,18. Em terceiro lugar, a expressão de TLR em células do estroma de nódulos linfáticos sugerem que as células do estroma são susceptíveis ao sinal obtido a partir de uma infecção ou de auto-moléculas libertado durante a lesão de tecidos. De facto, o tratamento de células do estroma de nódulos linfáticos com o ligando de TLR3 de poli (I: C) induz uma regulação positiva modesta de grande expressão de histocompatibilidade classe I e complexa regulação positiva da molécula co-inibidor PD-L1, mas não das moléculas de co-estimulação, resultando em mudanças dramáticas no tecido periférico antígenos expressão 19. Diversos grupos têm apontado células do estroma dos nódulos linfáticos expressar antígenos de tecidos periféricos e induzir tolerância das células T auto-reactivas 19,21-27. Portanto, a compreensão das interacções entre células do estroma dos nódulos linfáticos e outro re migratório ecélulas do linfonodo sident vai ajudar a encontrar novas moléculas alvo para permitir a ativação ou supressão de respostas imunes durante a inflamação. Portanto, é necessária a implementação da separação enzimática publicado do linfonodo. Protocolos previamente publicados usar diferentes combinações de digestão enzimática à base de colagenase com baixo esforço mecânico 6,19,20. No entanto, longas incubações com enzimas da digestão ou a combinação de diferentes tipos de enzima de digestão pode prejudicar várias moléculas de superfície necessárias para analisar o status de ativação e identificar novas células estromais de linfonodos. Dependendo do tipo de análise de células do estroma, o protocolo de ligação ou Fletcher protocolo pode ser mais adequado. No processo descrito, de uma digestão enzimática ligeiramente mais curto é combinado com a desagregação mecânica automatizado para minimizar a degradação da superfície marcador linfáticos viável células estromais nó. Este procedimento permite que o isolamento altamente reprodutível edistinção de nódulos linfáticos de populações de células do estroma com uma baixa variabilidade e de mais do que 95% de viabilidade. As células do estroma de nódulos linfáticos isolados de fresco pode ser usado directamente para a expressão do marcador de superfície, a análise de proteínas, e estudos de transcrição, bem como o estabelecimento de linhas de células estromais de realizar os ensaios funcionais in vitro.

<table border="0" cellpadding="0" cellspacing="0" width="959">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:231px;">LifeTechnologies-Gibco</td>
			<td style="width:119px;">41965-039</td>
			<td style="width:395px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FCS</td>
			<td style="width:231px;"></td>
			<td style="width:119px;"></td>
			<td>Final concentration 2%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:119px;">499609</td>
			<td>Final concentration 1.2 mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Collagenase IV</td>
			<td style="width:231px;">Worthington Biochemical Corporation</td>
			<td style="width:119px;"></td>
			<td>&#62;160 units per mg dry weight, use at final concentration of 1 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase D</td>
			<td style="width:231px;">Roche</td>
			<td style="width:119px;">11088882001</td>
			<td>use at 3.5 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNAse I</td>
			<td style="width:231px;">Roche&#160;</td>
			<td style="width:119px;">11284932001</td>
			<td>use at 40 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">stiring magnets</td>
			<td style="width:231px;">FAUST</td>
			<td style="width:119px;"></td>
			<td>5 mm long-2 mm &#248;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polystyren Round-Bottom Tubes 5ml</td>
			<td style="width:231px;">Falcon-BD Bioscience</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Magnetic stirrer with heating funktion</td>
			<td style="width:231px;">IKA-RCT-standard</td>
			<td style="width:119px;">9720250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petridishes 100 mm, sterile</td>
			<td style="width:231px;">TPP</td>
			<td style="width:119px;">6223201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25G needles</td>
			<td style="width:231px;">Terumo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD45 Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 30-F11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD11c Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone N418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse Podoplanin Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 8.1.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD31 Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone MEC13.3</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;"> 
			Eppendorf Xplorer plus, Multichannel</td>
			<td style="width:231px;">Eppendorf</td>
			<td style="width:119px;">4861 000.821/830</td>
			<td>1.250 &#181;l max. volume</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD140a</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone APA5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD80</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 16-10A1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD40</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 1C10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse I-Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone AF6-120.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD274 (PD-L1)</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 10F.9G2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LIVE/DEAD Fixable Near-IR Dead Cell stain kit</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:119px;">L10119</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

O presente protocolo é um protocolo de digestão modificada publicada por ligação et al., 2007 6 com um menor tempo de digestão (45 minutos no máximo), devido à desagregação mecânica com uma pipeta multicanal automatizado. Além disso, o processo é mais padronizado, minimiza a degradação de marcadores de superfície em diferentes células do estroma de nódulos linfáticos e permite a manipulação de mais de uma amostra ao mesmo tempo. Colagenase IV e colagenase D em links de protocolo 6 e protocolo atual ou colagenase P e Dispase em protocolo Fletchers 13 manter linfonodo estromal viabilidade das células e poupar a digestão de vários marcadores de superfície, incluindo CD45, gp38 e CD31. Para testar os nós de protocolo, axilar, braquial e linfáticos inguinal atuais foram removidos e digerido para isolar suas células estromais. Análise de CD45, gp38 e CD31 demonstraram a presença de expressão de TRC, LEC, BEC e DN células isoladas a partir de ratinhos C57BL / 6 mgelo (Figura 1, a estratégia de gating). O esforço mecânico pode reduzir a viabilidade das células linfáticas nó do estroma durante a digestão. Comparando-se a viabilidade de subpopulações isoladas de células do estroma, utilizando os três protocolos diferentes, verificou-se que a viabilidade foi superior a 95% no protocolo de Fletcher, mas inferior para o protocolo atual e protocolo Link (Figura 2A), embora o protocolo atual show melhor sobrevivência na subpopulação BEC então Link protocolo. O número total de células recuperado pós digestão do linfonodo subpopulações de células do estroma foi ligeiramente superior no protocolo atual em relação ao protocolo Link e protocolo Fletcher (Figura 2A). Estes resultados demonstram que o protocolo atual mantém a viabilidade das células estromais de linfonodos semelhantes aos protocolos publicados. As principais diferenças entre os três protocolos estão listados na Tabela 1. Marcadores de superfície de um<html> re necessária para caracterizar linfáticos células estromais nó por citometria de fluxo e isolá-las por separação de células. TRCs e LEC isolado via atual, os protocolos de ligação e Fletcher foram comparados por IA b, CD140a, CD80, PD-L1 e expressão de CD40. Após isolar células do estroma por todos os três protocolos, encontramos a expressão de IA b, CD80, CD140a, PD-L1, e CD40 foi maior após a digestão com CP e LP em ambos os TRCs e LEC (Figura 2B). Estes resultados sugerem que a degradação de algumas moléculas da superfície com colagenase IV e D são menos forte do que com colagenase e Dispase P. Tomados em conjunto, o protocolo atual inclui um curto digestão combinado com desagregação mecânica automatizada para minimizar a superfície marcador degradação dos linfáticos viável células estromais nó. &quot;/&gt; Figura 1 nós TRC, as células LEC, BEC, e DN coloração, gating, e quantificação. Linfáticos de camundongos C57BL / 6 foram digeridos seguindo o protocolo atual e corados com CD45, gp38 e CD31, Live / Dead to definir TRC, LEC, BEC e DN células por citometria de fluxo. Os dados mostram resultados representativos da coloração. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51803/51803fig1highres.jpg" target="_blank">Clique aqui para ver a imagem ampliada.</a> <img alt="Figura 2" fo:content-width="5in" src="/files/ftp_upload/51803/51803fig2highres.jpg" width="500" /> Figura 2 Viabilidade e superfície expressão do marcador de células estromais de linfonodos. Linfonodos de camundongos C57BL / 6 foram digeridos após a atual (PB), do protocolo de Fletcher (FP) e corados com CD45, gp38 e CD31 (LP) Link e definir TRC, LEC, BEC e DN células por citometria de fluxo. A) Viabildade e número total de células de todos os subpopulações de células dos linfonodos estromal após coloração Live / Dead (n ≥ 5, dados agrupados de dois experimentos independentes, barras representam SEM). B) Análise de expressão de superfície de IA b (APC-Cy7), CD140a (PerCP-Cy5.5), CD80 (PerCP-Cy5.5), PD-L1 (PE-Cy7) e CD40 (PE-Cy7) em TRCs e LEC (n = 6 para FP e n = 11 para LP e CP , dados agrupados de dois experimentos independentes, barras representam SEM). Geométricas das IFM valores auto-fluorescência para LEC-APC-Cy7: 324 ± 97, LEC-PECy7: 163 ± 82,5, LEC-PerCpCy5.5: 156 ± 36, TRC-APC-Cy7: 349 ± 152, TRC-PECy7: 117 ± 54, TRC-PerCP-Cy5.5: 121 ± 45. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51803/51803fig2highres.jpg" target="_blank">Clique aqui para ver a imagem ampliada.</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Atual Protocol-Enzimas </td><td> Protocolo atual </td><td> Protocolo de Ligação de enzimas </td><td> Protocolo de ligação </td><td> FLetcher Protocolo de enzimas </td><td> -Protocolo Fletcher </td></tr><tr><td> Colagenase IV, DNase I </td><td> 30 min, 37 ° C, sob agitação </td><td> Colagenase IV, DNAse I </td><td> 30 min, 37 ° C </td><td> Colagenase P, Dispase, DNAse I </td><td> 20 min, 37 ° C, 5 min cada inverter </td></tr><tr><td> Colagenase D, Dnase I </td><td> 5 min, 37 ° C, sob agitação, ressuspender </td><td> Colagenase D, DNAse I </td><td> 20 min, 37 ° C </td><td> Colagenase P, Dispase, DNAse I </td><td> 10 min, 37 ° C, 30 seg ressuspender </td></tr><tr><td></td><td> 10 min, 37 ° C, sob agitação </td><td> Colagenase D, DNAse I </td><td> 37 ° C, ressuspender a cada 10 minutos até que a digestão completa </td><td> Colagenase P, Dispase, DNAse I </td><td> 37 ° C, ressuspender a cada 5 min, até a digestão completa </td></tr><tr><td></td><td> ressuspensão mecânico automatizado </td><td></td><td></td><td></td><td></td></tr></tbody></table> Tabela 1. resumos curtos de CP, LP, enzimas PF usado e protocolos.

Watch this Scientific Journal Video about Isolation of Murine Lymph Node Stromal Cells at JoVE.com

Isolation of Murine Lymph Node Stromal Cells

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 ...

isolation-of-murine-lymph-node-stromal-cells

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

31.0K visualizações.  University of Basel and University Hospital Basel. 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. 

Vídeo: Isolation of Murine Lymph Node Stromal Cells

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Microscopia intravital do linfonodo inguinal

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Imunologia e Infecção

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Ex vivo de imagens de células T em fatias Node murino linfáticos com Widefield e microscópios confocal

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Murine Superficial Lymph Node Surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

Cirurgia Nó murino superficial linfático

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Imagem intravital do Linfonodo Popliteal Rato

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

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 ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

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 ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Isolamento de células mielóides de rato Pele e Drenagem Linfonodo Seguindo intradérmica imunização com vivos atenuados<em&gt; Plasmodium</em&gt; Esporozoítos

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

Coleta e processamento de linfonodos de animais de grandes porte para a análise do RNA: preparando-se para estudos de linfonodo Transcriptomic de espécies de animais grandes

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

Determinação de subconjuntos de regulação de células T no timo murino, pancreático drenagem do linfonodo e baço usando citometria de fluxo

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...