This study has been supported by the Collaborative Research Centre CRC TR128 "Initiating/Effector versus Regulatory Mechanisms in Multiple Sclerosis-Progress towards Tackling the Disease" (Project A9 to H.W. and C.C.G., project B1 to N.S.).

1. Cultura de células de cérebro humano Células Endoteliais Microvasculares (HBMEC) <ol><li> Revestimento de frascos de cultura de células <ol><li> Para preparar a solução de fibronectina, adicionar 10 mL de PBS para um tubo de centrífuga de 15 mL. Adicionar 150 mL de fibronectina e misture bem. </li><li> Para cobrir o fundo de um frasco de cultura de células T-25, adicionar 2 ml de solução de fibronectina. Incubar o balão de cultura de células durante pelo menos 3 h a 37 ° C na incubadora. Revestidas com fibronectina frascos podem ser armazenados durante 2 semanas a 37 ° C / 5% de CO 2. </li></ol></li><li> Sementeira e cultura de células de HBMEC <ol><li> Aspirar solução de fibronectina a partir do fundo do frasco de cultura de célula. Adicionar 7,2 x 10 4 HBMEC / cm² suspenso em 6 ml de meio de ECM-b (= ECM-b suplementado com 5% de soro fetal bovino, 1% de penicilina / estreptomicina e 1% de suplemento de crescimento de células endoteliais). Incubar a 37 ° C / 5% de CO 2. Verifique o crescimento de células diariamente usando um microscópio. </li><li> Mudar o meio cada 3 dias.Colheita ou células divididas, quando HBMEC atingir aproximadamente 80% de confluência. HBMEC deve ser usado entre a passagem 1 e 15 para evitar a perda de propriedades fisiológicas. </li></ol></li><li> Colheita HBMEC. <ol><li> Prepare Accutase solução por mistura de Accutase (1x) com PBS numa proporção de 1: 1. Manter Accutase solução a 37 ° C em um banho de água até à sua utilização. </li><li> Transferência médio ECM-b a partir do balão de cultura de células para um tubo de centrífuga de 15 mL. Lavar HBMEC por adição de 5 mL de PBS para o fundo do frasco de cultura de células. Aspirar PBS e repetir o passo de lavagem mais duas vezes. </li><li> Adicionar a solução de 2 ml pré-aquecido Accutase. Incubar a 37 ° C durante 2 min. Posteriormente, HBMEC são re-suspensos batendo firmemente o frasco de cultura de células várias vezes. destacamento das células é controlada usando um microscópio </li><li> O ECM-b-forma previamente armazenado num tubo de 15 ml é adicionado de volta ao frasco de cultura de células, logo que HBMEC começar a separar. Lavar o fundo do frasco repetidamente até que a maioria são HBMECre-suspensa. </li><li> Transferir a suspens celular para um tubo de centrífuga de 15 mL. Centrifugar a 300 xg durante 10 min à temperatura ambiente. Elimine o sobrenadante e as células em 1 mL de meio de ECM-b re-suspender. Contar as células e dilui-se a suspensão de células para se conseguir uma concentração final de 3 x 10 5 mL HBMEC por meio de ECM-b. </li></ol></li></ol> 2. Preparação do celular Cultura Insere <ol><li> Revestimento de inserções de cultura de células Nota importante: Evite tocar a membrana das inserções de cultura de células. <ol><li> Adicionar 100 de soluo de fibronectina (ver 1.1.1) de cada inserção de cultura de células (Figura 1A) e uma cavidade de uma placa de 96 poços de fundo plano (controlo óptico bem). Incuba-se durante pelo menos 3 h a 37 ° C. Depois da solução de incubação aspirado fibronectina. </li><li> Adicionar 100 mL HBMEC suspensão para as inserções de cultura de células e o controlo óptico bem. Adicionar 600 ul de ECM-b meio para o compartimento inferior da célulainserções cultura. Incubar durante 3 - 4 dias a 37 ° C / 5% de CO2 até que a integridade da barreira (Figura 1B) é atingido, verificar o crescimento celular por avaliação microscópica do HBMEC no controlo óptico bem. Nota: O crescimento celular para além de quatro dias não é recomendado. </li><li> Opcional: Para imitar as condições inflamatórias aspirar o meio a partir do compartimento inferior e substituí-la por ECM-b meio suplementado com 500 U / mL de IFN-γ / TNF-α 24 h antes do ensaio de migração. </li></ol></li></ol> 3. Controle de Qualidade com azul Evans no dia do ensaio Transmigração <ol><li> Preparação de solução de azul de Evans <ol><li> Para preparar PBS / B27 solução mistura de 10 mL de PBS com suplemento B27 200 ul utilizando um tubo de 15 ml de centrífuga. Diluir solução mãe de azul de Evans (20 mg / mL PBS) 1: 1000 com PBS / B27. </li></ol></li><li> ensaio de permeabilidade de azul de Evans <ol><li> Aspirar o meio do compartimento inferior seguido pelo compartimento superiorde uma inserção de cultura de célula contendo uma monocamada confluente HBMEC. Adicionar 100 mL Evans solução azul para a inserção de cultura de célula. </li><li> Adicionar 600 de PBS / B27 para o compartimento inferior e incubar durante 60 min a 37 ° C / 5% de CO 2. Cuidadosamente remover a inserção de cultura de célula usando uma pinça. </li></ol></li><li> medição de azul de Evans <ol><li> Remover PBS / B27 a partir do compartimento inferior e transferir 100 ul cada um de dois pos de uma polistirol preto placa de 96 poços de fundo plano. Inserir a placa num leitor de placas Tecan Pro Infinito M200 e determinar-z posição óptima. </li><li> excitação medida de azul de Evans, utilizando respectivas configurações (por exemplo: de excitação: 620 nm, emissão: 680 nm, largura de banda de excitação: 9 nm, largura de banda de emissão: 20 nm, realce 175x, 25 flashes, tempo de integração: 20 mS). </li><li> Para determinar as funções de barreira HBMEC comparar os dados adquiridos para uma curva padrão representando Evans permeação azul através HBMEC em diferentes pontos de tempo após a sementeing células (Figura 1B, à direita). </li></ol></li></ol> 4. Migração Assay <ol><li> Preparação de células mononucleares de sangue periférico (PBMC). <ol><li> Adicionar 10 mL de meio RPMI para um tubo de centrífuga de 15 ml e adiciona-se 200 uL suplemento B27. Contagem de células PBMC e de centrifugação a 300 xg durante 5 min. Re-suspender as PBMC a uma concentração final de 5 x 10 6 culas / mL de RPMI / B27. </li></ol></li><li> Defina-se o ensaio de migração <ol><li> Meio aspirado a partir do compartimento inferior seguido pelo compartimento superior de inserções de cultura de células contendo monocamadas confluentes HBMEC (Figura 1A). Por doador, adiciona-se 100 uL de suspensão de PBMC de cada para as inserções de cultura de células e também a um poço de uma placa de 24 poços por (in vitro de controlo). </li><li> Adicionar 600? L de RPMI / B27 para o compartimento inferior das inserções de cultura de células e 500 ul para o PBMC do controlo in vitro e incubar 6 h a 37 ° C / 5% de CO 2. </li&gt; </ol></li><li> Colheita de PBMC migrado <ol><li> Retire a inserção de cultura de célula usando uma pinça e lavar cuidadosamente a parte inferior com 400 uL de PBS sem tocar na membrana. Descartar a inserção de cultura de célula. </li><li> Adicionar 20 ul fluorosferas contagem de fluxo (cerca de 1000 esferas / uL) para o compartimento inferior da cultura celular inserir, bem como para o controlo in vitro e misturar bem. Transferir 1 ml da resultante suspensão de PBMC para tubos de citometria de fluxo. </li></ol></li></ol> 5. Citometria de Fluxo <ol><li> Preparação de amostra <ol><li> Centrífuga PBMC a 300 xg durante 5 min à temperatura ambiente. </li><li> Preparar a soluo de anticorpo através da adição de anticorpos fluorocromo-conjugados com 100 uL de tampão de citometria de fluxo (PBS / BSA a 1% / EDTA a 2 mM) por amostra. Para os resultados apresentados abaixo 1 uL de CD4-FITC, um μLCD3-PerCP / Cy5.5, 1 uL de CD56-PC7, 1 uL CD8-A700, e 1 uL de CD16-A750 foram usadas por amostra. </li><li> Re-suspender PBMC em 100 mL da solução de anticorpo e incubar durante 30 min a 4 ° C. </li><li> Adicionar 250 uL de tampão de citometria de fluxo e centrifugar a 300 xg durante 5 min. </li></ol></li><li> aquisição amostra <ol><li> Re-suspender as PBMC na quantidade necessária (varia de acordo com o citetro de fluxo utilizado) de tampão de citometria de fluxo. </li><li> Adquirir manchado de PBMC utilizando um citómetro de fluxo com um detector activa entre 525 e 700 nm comprimento de onda para detectar fluorosferas contagem de fluxo (excitação 488 nm, emissão a 525-700 nm). (Os passos seguintes são um exemplo, se um fluxo Gallios citómetro operado com o software Kaluza G é usada:. (1) Iniciar o computador (2) Quando o sistema de operação está completamente carregado, iniciar o citómetro de fluxo por prensagem a &quot;citómetro no&quot; botão . (3) Coloque o respectivo protocolo de aquisição, premindo o botão &quot;protocolo aberto&quot;. (4) Escolha o protocolo necessário e selecione &quot;abrir&quot;. (5) Duplicar o protocolo para cada amostra, clicando com o direitobotão do mouse sobre o protocolo visível no carrossel virtual e um clique esquerdo no campo &quot;duplicado&quot;. (6) Rotular cada amostra na lista de amostra. (7) Transferir as amostras para as posições indicadas do carrossel e iniciar a aquisição.) </li></ol></li><li> análise de amostras <ol><li> Abrir fluxo resultante citometria de dados usando o respectivo software. Determinar o número de sub-populações de interesse para PBMC transmigradas, bem como células in vitro a partir de poços de controlo e fluir fluorosferas contagem usando o respectivo software de análise. (Um exemplo da estratégia de intermitcia é dada na parte resultados (Figura 1 C: Para analisar a transmigração de subconjuntos de culas NK, seleccionar primeiro os linfócitos em um canal de dispersão para um lado (SSC) em relação ao canal de dispersão FSC) trama (linfócitos são. então exibidos num CD3 contra CD56 trama e CD56 + CD3 -. As células NK são seleccionados para distinguir entre os subconjuntos de células NK, células NK são exibidas emum contra CD56 CD16 CD56 CD16 trama e brilhante dim / - células NK, bem como CD56 CD16 + são seleccionadas dim. Além disso, fluorosferas contagem de fluxo são seleccionados a partir de um FSC SSC contra trama e posteriormente apresentado numa trama de um canal com uma emissão entre 525 e 700 nm versus tempo para determinar o seu número.) </li><li> Para calcular o número total de células de cada amostra, normalizar o número de células detectadas usando fluorosferas contagem de fluxo: <img alt="Equação" src="/files/ftp_upload/55390/55390eq1.jpg" /></li><li> Determinar a percentagem de células que migraram como a razão entre células que migraram totais e de células totais no controlo in vitro. </li></ol></li></ol>

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: A.S.-M. and U.B. have no financial disclosures. T. S.-H. received travel and conference expenses from Biogen. N.S. received speaker and advisory board honoraria from Biogen and Novartis Pharma, as well as travel expenses from Biogen. H.W. received compensation for serving on Scientific Advisory Boards/Steering Committees for Bayer Healthcare, Biogen, Merck Serono, Novartis, and Sanofi-Genzyme. He also received speaker honoraria and travel support from Bayer Vital GmbH, Bayer Schering AG, Biogen, CSL Behring, Fresenius Medical Care, Glaxo Smith Kline, GW Pharmaceuticals, Lundbeck, Merck Serono, Omniamed, Novartis, and Sanofi-Genzyme. He received compensation as a consultant from Biogen, Merck Serono, Novartis, and Sanofi-Genzyme. H.W. received research support from Bayer Vital, Biogen, Genzyme, Merck Serono, Novartis, Sanofi-Aventis Germany, and Sanofi US. C.C.G. received speaker honoraria and travel expenses for attending meetings from Genzyme, Novartis Pharma GmbH, and Bayer Health Care.

Aqui apresentamos uma técnica para investigar a transmigração de linfócitos através da barreira sangue-cérebro humano. A análise in vitro de migração de linfócitos para o SNC é importante para estudar processos básicos de extravasamento de linfitos, potenciais alterações relacionadas com a doença, e de novas abordagens terapêuticas. Várias modificações do modelo de barreira sangue-cérebro são possíveis. Por exemplo, as células do compartimento superio...

migração de linfócitos a partir do sangue para os tecidos é crucial para a vigilância imunitária. Uma sequência de interacções moleculares específicos assegura extravasamento local específico para o intestino delgado, de pele, nódulos linfáticos, o sistema nervoso central (CNS), e outros tecidos 1. Alterações na migração de linfócitos estão envolvidos na patofisiologia de uma série de doenças ampla disseminação 2. Migração para o SNC imuno-privilegiado é fortemente regulada e, consequentemente, alterações deste processo estão envolvidos em doenças relacionadas com o SNC, como encefalomielite 3, neuromielite óptica, acidente vascular cerebral, e esclerose múltipla (MS), 2, 4, 5, 6, 7. Portanto, é importante estudar o extravasamento de linfócitos para melhor compreender a fisiopatologia da doença e desenvolver ferramentas para uma<html> melhoramento da doença de carga 8, 9, 10, 11, 12. Os linfócitos migra para o SNC através de rotas distintas. Extravasamento através de vénulas pós-capilares para o espaço subaracnóide através da barreira sangue-fluido cerebrospinal dentro do plexo coróide e através da barreira sangue-cérebro ter sido descrito um, 13, 14, 15. Migração através da barreira sangue-cérebro é conduzida pela interacção de linfócitos com células endoteliais 14. Em contraste com as células endoteliais na periferia, as células endoteliais do SNC expressar quantidades elevadas de moléculas de juno apertadas, assim limitar rigorosamente a quantidade de células e proteínas capazes de atravessar a barreira sangue-cérebrolass = &quot;xref&quot;&gt; 16. Inflamação resulta em afrouxamento de junções apertadas e induz a expressão de moléculas de adesão; assim, aumentando a migração de linfócitos para o SNC 1, 17, 18. Extravasamento através da barreira sangue-cérebro é um processo de múltiplos passos. Linfócitos do tirante para as células endoteliais e, em seguida, rolar ao longo do endotélio em um processo mediado principalmente por selectinas 1, 15. Posteriormente, as interacções entre as quimioquinas segregadas pelo endotélio e os respectivos receptores de quimiocinas expressas em linfócitos induzir mudanças conformacionais de integrinas, promovendo assim a adesão firme para as células endoteliais 1. Finalmente, quer linfócitos de rastreamento ao longo da barreira endotelial contra o fluxo de sangue antes transmigrando para o espaço perivascular, ou parar imediatamente e directamente transmigrate no local da empresa de aderência 1, 19, 20. Todos estes passos de extravasamento de linfitos pode ser analisada in vitro utilizando técnicas distintas 21. Microscopia de lapso de tempo vídeo é usado para estudar o tethering inicial e rolando 15. Os ensaios de adesão fornecem informações detalhadas sobre a prisão firme endoteliais barreiras 22. Os ensaios de transmigração como demonstrado aqui permitem a análise de transmigração de células imunes 21, 23, 24, 25, 26, 27, 28, 29. Usando o modelo humano in vitro barreira hematoencefálica, que recentemente poderia mostrar que uma maior migrAtory capacidade de CD56 CD16 brilhante dim / - células NK em comparação com o seu CD56 dim CD16 + homólogos foi reflectida por uma predominância deste subconjunto de células NK no compartimento 21 intratecal. Assim, a instalação experimental parece ser adequado para mimetizar a situação in vivo.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td>without CaCl2 or MgCl2</td>
		</tr>
		<tr>
			<td>Fibronectin 1mg/mL</td>
			<td>Sigma</td>
			<td>F1141-5MG</td>
			<td>from bovine plasma</td>
		</tr>
		<tr>
			<td>T-25 cell culture flask</td>
			<td>Greiner BioOne</td>
			<td>690160</td>
			<td></td>
		</tr>
		<tr>
			<td>HBMEC</td>
			<td>ScienCell</td>
			<td>1000</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Pelobiotech</td>
			<td>PB-H-6023</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma</td>
			<td>A6964-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>ECM-b</td>
			<td>ScienCell</td>
			<td>1001-b</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>ScienCell</td>
			<td>1001-b</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>ScienCell</td>
			<td>1001-b</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial cell growth supplement</td>
			<td>ScienCell</td>
			<td>1001-b</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell</td>
			<td>Corning</td>
			<td>3472</td>
			<td>clear, 6.5mm diameter, 3.0&#181;m pore size</td>
		</tr>
		<tr>
			<td>96-well flat bottom plate</td>
			<td>Corning</td>
			<td>3596</td>
			<td></td>
		</tr>
		<tr>
			<td>Evans blue</td>
			<td>Sigma</td>
			<td>E2129-10G</td>
			<td>stock solution: 1 g/50 mL PBS</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>50x concentrated</td>
		</tr>
		<tr>
			<td>Infinite M200Pro</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well black flat bottom plate</td>
			<td>Greiner BioOne</td>
			<td>675086</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well plate</td>
			<td>Corning</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco</td>
			<td>61870-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Count Fluorospheres</td>
			<td>Beckman Coulter</td>
			<td>7547053</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-EDTA</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Gallios 10-color flow cytometer</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kaluza 1.5a</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TNF-&#945;</td>
			<td>Peprotech</td>
			<td>300-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>IFN-&#947;</td>
			<td>Peprotech</td>
			<td>300-02</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3-PerCP/Cy5.5</td>
			<td>Biolegend</td>
			<td>300430</td>
			<td>clone UCHT1</td>
		</tr>
		<tr>
			<td>CD56-PC7</td>
			<td>Beckman Coulter</td>
			<td>A21692</td>
			<td>clone N901</td>
		</tr>
		<tr>
			<td>CD16-A750</td>
			<td>Beckman Coulter</td>
			<td>A66330</td>
			<td>clone 3G8</td>
		</tr>
		<tr>
			<td>CD4-FITC</td>
			<td>Biolegend</td>
			<td>300506</td>
			<td>clone RPA-T4</td>
		</tr>
		<tr>
			<td>CD8-A700</td>
			<td>Beckman Coulter</td>
			<td>A66332</td>
			<td>clone B9.11</td>
		</tr>
	</tbody>
</table>

analysis of lymphocyte extravasation using an in vitro model of the human blood-brain barrier

Linfócitos extravasão para o sistema nervoso central (SNC) é crítica para a vigilância imunitária. alterações relacionadas com a doença de extravasamento de linfócitos pode resultar em alterações fisiopatológicas no SNC. Assim, investigação sobre a migração de linfócitos para o SNC é importante para entender as doenças inflamatórias do sistema nervoso central e o desenvolvimento de novas abordagens de terapia. Aqui apresenta-se um modelo in vitro da barreira sangue-cérebro humano para estudar o extravasamento de linfitos. células endoteliais humanas microvasculares cerebrais (HBMEC) são cultivadas em confluently um tereftalato de polietileno poroso Transwell Insert para imitar o endotélio da barreira sangue-cérebro. a função de barreira é validado por zonula occludens imuno-histoquímica, a resistência eléctrica transendotelial (TEER) medidas bem como a análise de Evans permeação azul. Este modelo permite a investigação da diapedese de subconjuntos de linfócitos raras, tais como CD56 CD16 brilhante dim / - células NK. Furthermminério, os efeitos de outras células, citocinas e quimiocinas, alterações relacionadas com a doença, e regimes de tratamento diferentes na capacidade migratória de linfócitos podem ser estudados. Finalmente, o impacto de estímulos inflamatórios, bem como diferentes regimes de tratamento sobre a barreira endotelial pode ser analisado.

Os resultados representativos, mostrando a transmigração de células NK e subconjuntos de células T utilizando o modelo de barreira sangue-cérebro humano (Figura 1A) são mostrados. A integridade da monocamada HBMEC foi validado por coloração da molécula de junção estanque ZO-1, a resistência eléctrica medições transendotelial (TEER), e Evans permeação azul (Figura 1B). Seguindo 3 - 4 dias de cultura HBMEC expressa a molécula de junção ...

Watch this Scientific Journal Video about Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier at JoVE.com

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Análise de Linfócitos extravasamento Usando um<em&gt; In Vitro</em&gt; Modelo do Sangue-Cérebro humano Barreira

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

analysis-lymphocyte-extravasation-using-an-vitro-model-human-blood

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Immunology and Infection

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

20.3K visualizações.  University Hospital Münster. Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro. 

Vídeo: Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Isolamento T de linfócitos humanos, Cultura e Análise da Migração In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Imunologia e Infecção

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

Ponta de dedo Metodologia de amostragem de sangue para a determinação da apoptose induzida pelo exercício de Linfócitos

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Humano In Vitro Supressão como ferramenta de triagem para o reconhecimento de um Estado precoce de desequilíbrio imunológico

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Um<em&gt; In vitro</em&gt; Modelo para estudar a heterogeneidade de diferenciação de macrófagos humanos e Polarização

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Uma<em&gt; In vitro</em&gt; Modelo para estudar respostas imunes de sangue periférico humano células mononucleares para Vírus Respiratório Sincicial Humano Infecção

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Investigando os efeitos dos probióticos sobre pneumocócica Colonização Usando um<em&gt; In Vitro</em&gt; Adesão Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

Desenvolvimento de um<em&gt; In vitro</em&gt; Sistema modelo para estudar a interacção de<em&gt; Equus</em&gt;<em&gt; Caballus</em&gt; IgE ao seu receptor de elevada afinidade FceRI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Isolamento de linfócitos da pele humana para análise fenotípica e<em&gt; Ex Vivo</em&gt; Cultura de Células

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

A<em&gt; In Vitro</em&gt; Modelo da barreira sangue-cérebro que usa espectroscopia de impedância: Um foco na interação celular T Cell-endotelial

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran aumenta a eficiência de Lentivirus transdução das células NK primário murino e humano

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...