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. Cultivo celular de cerebro humano Las células endoteliales microvasculares (HBMEC) <ol><li> Recubrimiento de matraces de cultivo celular <ol><li> Para preparar la solución de fibronectina, añadir 10 ml de PBS a un tubo de centrífuga de 15 ml. Añadir 150! L fibronectina y mezclar bien. </li><li> Para cubrir la parte inferior de un frasco de cultivo de células T-25 Añadir 2 ml de la solución de fibronectina. Incubar el matraz de cultivo de células durante al menos 3 h a 37 ° C en la incubadora. Matraces recubiertos de fibronectina se pueden almacenar durante 2 semanas a 37 ° C / 5% de CO2. </li></ol></li><li> La siembra y cultivo de células de HBMEC <ol><li> solución de fibronectina Aspirar desde el fondo del matraz de cultivo celular. Añadir 7,2 x 10 4 HBMEC / cm² en suspensión en 6 ml ECM-b medio (= ECM-b suplementado con 5% de suero bovino fetal, 1% de penicilina / estreptomicina y 1% de suplemento de crecimiento endotelial celular). Incubar a 37 ° C / 5% de CO2. Compruebe el crecimiento celular diariamente utilizando un microscopio. </li><li> Cambiar el medio cada 3 días.Recoger o de células de división, cuando HBMEC alcanzan aproximadamente 80% de confluencia. HBMEC se debe utilizar entre el paso 1 y 15 para evitar la pérdida de propiedades fisiológicas. </li></ol></li><li> Cosecha HBMEC. <ol><li> Preparar Accutase solución mezclando Accutase (1x) con PBS en una proporción de 1: 1. Mantenga Accutase solución a 37 ° C en un baño de agua hasta su uso posterior. </li><li> Transferencia ECM-b medio del matraz de cultivo celular a un tubo de centrífuga de 15 ml. Lavar HBMEC mediante la adición de 5 ml de PBS a la parte inferior del frasco de cultivo celular. Aspirar PBS y repetir la etapa de lavado dos veces más. </li><li> Añadir 2 ml de solución pre-calentado Accutase. Incubar a 37 ° C durante 2 min. Después, HBMEC se resuspenden tocando firmemente el matraz de cultivo de células varias veces. desprendimiento de células se controla mediante un microscopio </li><li> El ECM-b-medio almacenado previamente en un tubo de 15 ml se añadió de nuevo al matraz de cultivo celular tan pronto como se inicia HBMEC a separar. Enjuague el fondo del matraz varias veces hasta que la mayoría son HBMECre-suspendido. </li><li> Transferir la suspensión celular a un tubo de centrífuga de 15 ml. Centrifugar a 300 xg durante 10 min a temperatura ambiente. Desechar el sobrenadante y las células en 1 ml ECM-b medio resuspender. Recuento de células y diluir la suspensión de células para alcanzar una concentración final de 3 x 10 5 HBMEC por ml ECM-b medio. </li></ol></li></ol> 2. Preparación de la Célula de Cultura Inserts <ol><li> Recubrimiento de insertos de cultivo celular Nota importante: Evitar tocar la membrana de los insertos de cultivo celular. <ol><li> Añadir 100! L solución de fibronectina (véase 1.1.1) para cada inserto de cultivo celular (Figura 1A) y un pocillo de una placa de 96 pocillos de fondo plano (control óptico también). Incubar durante al menos 3 h a 37 ° C. Después de solución de incubación aspirado de fibronectina. </li><li> Añadir 100 ml HBMEC suspensión a los insertos de cultivo celular y el control óptico también. Añadir 600 l ECM-b medio para el compartimiento inferior de la célulainsertos de cultivo. Incubar durante 3 - 4 días a 37 ° C / 5% de CO 2 hasta integridad de la barrera (Figura 1B) se alcanza, comprobar el crecimiento celular por evaluación microscópica de la HBMEC en el control óptico también. Nota: No se recomienda el crecimiento celular más allá de cuatro días. </li><li> Opcional: Para imitar las condiciones inflamatorias Aspirar el medio desde el compartimiento inferior y reemplazarlo con ECM-b medio suplementado con 500 U / ml de IFN-γ / TNF-α 24 h antes el ensayo de migración. </li></ol></li></ol> 3. Control de Calidad con azul de Evans en el Día del ensayo de transmigración <ol><li> Preparación de solución de azul de Evans <ol><li> Para preparar PBS / B27 mezcla de solución 10 ml de PBS con 200 l suplemento B27 usando un tubo de centrífuga de 15 ml. Diluir la solución madre de azul de Evans (20 mg / ml PBS) 1: 1000 con PBS / B27. </li></ol></li><li> ensayo de permeabilidad azul de Evans <ol><li> Aspirar el medio desde el compartimiento inferior seguido por el compartimiento superiorde un inserto de cultivo celular que contiene un confluente HBMEC monocapa. Añadir 100! L de solución de azul de Evans al inserto de cultivo celular. </li><li> Añadir 600! L de PBS / B27 para el compartimiento inferior y se incuba durante 60 min a 37 ° C / 5% de CO2. Retire cuidadosamente la inserción de cultivo celular utilizando fórceps. </li></ol></li><li> medición de azul de Evans <ol><li> Retirar PBS / B27 desde el compartimiento inferior y transferir 100! L cada uno a dos pocillos de una poliestirol negro placa de 96 pocillos de fondo plano. Inserte la placa en un lector de placa Tecan Pro Infinito M200 y determinar z-posición óptima. </li><li> Medida de excitación de azul usando ajustes respectivos Evans (por ejemplo: excitación: 620 nm, emisión: 680 nm, ancho de banda de excitación: 9 nm, ancho de banda de emisión: 20 nm, la mejora 175x, 25 parpadea, el tiempo de integración: 20 mu s). </li><li> Para determinar las funciones de barrera HBMEC comparar datos adquiridos con una curva patrón que representa evans permeación azul a través HBMEC en diferentes puntos de tiempo después de semillacélulas ING (Figura 1B, derecha). </li></ol></li></ol> 4. Ensayo de migración <ol><li> Preparación de células mononucleares de sangre periférica (PBMC). <ol><li> Añadir 10 ml de RPMI en un tubo de centrífuga de 15 ml y añadir 200 l suplemento B27. Recuento de células PBMC y centrifugar a 300 xg durante 5 min. Vuelva a suspender PBMC a una concentración final de 5 x 10 6 células / ml RPMI / B27. </li></ol></li><li> Puesta en marcha del ensayo de migración <ol><li> Medio Aspirar desde el compartimiento inferior seguido por el compartimiento superior de los insertos de cultivo de células que contienen confluente HBMEC monocapas (Figura 1A). Por donante añadir 100 l PBMC suspensión cada uno para los insertos de cultivo celular y también a un pocillo de una placa de 24 pocillos por (control in vitro). </li><li> Añadir 600! L RPMI / B27 para el compartimiento inferior de los insertos de cultivo celular y 500 l a la PBMC del control in vitro y se incuba 6 horas a 37 ° C / 5% de CO2. </li&gt; </ol></li><li> La recolección de migrada PBMC <ol><li> Extraer el inserto de cultivo celular utilizando fórceps y enjuagar cuidadosamente la parte inferior con 400 l de PBS sin tocar la membrana. Desechar el inserto de cultivo celular. </li><li> Añadir 20 l fluoroesferas de recuento de flujo (aproximadamente 1.000 cuentas / ml) al compartimento inferior del cultivo celular insertan así como para el control in vitro y se mezclan bien. Transferir 1 ml de resultante suspensión de PBMC a la citometría de flujo tubos. </li></ol></li></ol> 5. Citometría de Flujo <ol><li> preparación de la muestra <ol><li> Centrifugar PBMC a 300 xg durante 5 min a temperatura ambiente. </li><li> Preparar la solución de anticuerpo mediante la adición de anticuerpos conjugados con fluorocromo a 100! L de tampón citometría de flujo (/ EDTA mM PBS / 1% BSA 2) por muestra. Para los resultados presentados a continuación 1 l CD4-FITC, 1 μLCD3-PerCP / Cy5.5, 1 l CD56-PC7, 1 l CD8-A700, y 1 l CD16-A750 se utilizaron por muestra. </li><li> Resuspender PBMC en 100 l de la solución de anticuerpo y se incuba durante 30 min a 4 ° C. </li><li> Añadir 250! L de tampón de citometría de flujo y se centrifuga a 300 xg durante 5 min. </li></ol></li><li> la adquisición de muestras <ol><li> Vuelva a suspender PBMC en la cantidad requerida (varía dependiendo de la citómetro de flujo utilizado) de citometría de flujo de tampón. </li><li> Adquirir manchado PBMC usando un citómetro de flujo con un detector activo entre 525 y 700 nm de longitud de onda para detectar fluoroesferas de recuento de flujo (excitación 488 nm, emisión de 525 - 700 nm). (Los pasos siguientes son un ejemplo si se usa un flujo Gallios citómetro operado con software Kaluza G:. (1) Iniciar el equipo (2) Cuando el sistema operativo se ha cargado completamente, inicie el citómetro de flujo pulsando la tecla &quot;citómetro en&quot; botón . (3) Cargar el protocolo de adquisición respectiva pulsando el botón &quot;protocolo abierto&quot;. (4) seleccione el protocolo requerido y seleccione &quot;abrir&quot;. (5) Duplicar el protocolo para cada muestra haciendo clic con el derechobotón del ratón sobre el protocolo visible en el carrusel virtual y un clic izquierdo en el campo &quot;duplicado&quot;. (6) Etiqueta de cada muestra en la lista de muestras. (7) Transferir las muestras a las posiciones indicadas del carrusel y comenzar la adquisición.) </li></ol></li><li> análisis de las muestras <ol><li> Abrir flujo resultante citometría de datos utilizando el software respectivo. Determinar el número de subpoblaciones de interés para transmigrado PBMC así como las células de los pocillos de control in vitro y fluoroesferas de recuento de flujo usando el software de análisis respectiva. (Un ejemplo de la estrategia de gating se da en la parte resultados (Figura 1 C: Para analizar la transmigración de los subconjuntos de células NK, primero seleccionar linfocitos en un canal de dispersión lateral (SSC) frente hacia adelante canal de dispersión parcela (FSC) Los linfocitos son. a continuación, muestran en una CD3 frente trama CD56 y CD56 + CD3 -. se seleccionan las células NK Para distinguir entre subconjuntos de células NK, las células NK se muestran enuna parcela CD56 frente a CD16 y CD56 CD16 brillante dim / - las células NK, así como CD56 dim CD16 + se seleccionan. Además, fluoroesferas de recuento de flujo se seleccionan de una parcela FSC frente a SSC y, posteriormente, se muestra en una parcela de un canal con una emisión entre 525 y 700 nm frente al tiempo para determinar su número). </li><li> Para calcular el número total de células de cada muestra, normalizar el número detectado de las células usando fluoroesferas de recuento de flujo: <img alt="Ecuación" src="/files/ftp_upload/55390/55390eq1.jpg" /></li><li> Determinar el porcentaje de células migradas como la relación entre el total de células migradas y las células totales en el control 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.

Aquí presentamos una técnica para investigar la transmigración de los linfocitos a través de la barrera sangre-cerebro humano. El análisis in vitro de la migración de linfocitos al SNC es importante para estudiar los procesos básicos de la extravasación de linfocitos, posibles alteraciones relacionadas con la enfermedad, y los nuevos enfoques terapéuticos. Varias modificaciones del modelo de barrera hematoencefálica son posibles. Por ejemplo, las células del comp...

la migración de linfocitos de la sangre a los tejidos es crucial para la vigilancia inmune. Una secuencia de interacciones moleculares específicas asegura sitio extravasación específico en el intestino delgado, piel, ganglios linfáticos, el sistema nervioso central (SNC), y otros tejidos 1. Las alteraciones en la migración de linfocitos están implicados en la fisiopatología de una serie de enfermedades de amplia difusión 2. Migración en la inmuno-privilegiado CNS está estrechamente regulada y, en consecuencia alteraciones de este proceso están involucrados en las enfermedades relacionadas con el SNC como la encefalomielitis 3, neuromielitis óptica, derrame cerebral y la esclerosis múltiple (MS) 2, 4, 5, 6, 7. Por lo tanto, es importante estudiar la extravasación de linfocitos para entender mejor la fisiopatología de la enfermedad y el desarrollo de herramientas para una<html> mejoramiento del terreno de carga 8, 9, 10, 11, 12 enfermedad. Los linfocitos migran al SNC a través de rutas distintas. La extravasación a través de las vénulas postcapilares en el espacio subaracnoideo a través de la barrera sangre-líquido cefalorraquídeo dentro del plexo coroideo y través de la barrera sangre-cerebro se han descrito 1, 13, 14, 15. La migración a través de la barrera sangre-cerebro se lleva a cabo por la interacción de los linfocitos con las células endoteliales 14. En contraste a las células endoteliales en la periferia, las células endoteliales de la CNS expresan altas cantidades de moléculas de unión estrecha, con lo estrictamente limitando la cantidad de células y proteínas capaces de cruzar la barrera sangre-cerebrolass = &quot;xref&quot;&gt; 16. resultados inflamación en el aflojamiento de las uniones estrechas e induce la expresión de moléculas de adhesión; por lo tanto, la mejora de la migración de linfocitos en el SNC 1, 17, 18. La extravasación a través de la barrera sangre-cerebro es un proceso de múltiples pasos. Linfocitos de sujeción para las células endoteliales y luego rodar a lo largo del endotelio en un proceso mediado principalmente por las selectinas 1, 15. Posteriormente, las interacciones entre las quimiocinas secretadas por el endotelio y los respectivos receptores de quimioquinas expresados en los linfocitos inducen cambios conformacionales de las integrinas, promoviendo así la adhesión firme a las células endoteliales 1. Finalmente, los linfocitos o bien arrastre a lo largo de la barrera endotelial contra el flujo de sangre antes de transmigrando en el espacio perivascular, o detener inmediatamente y directamente transmigrate en el sitio de la firma de la adhesión 1, 19, 20. Todos estos pasos de extravasación de linfocitos pueden ser analizados in vitro usando técnicas distintas 21. Time-lapse microscopía de vídeo se utiliza para estudiar la inmovilización inicial y 15 de rodamiento. Ensayos de adhesión proporcionan información detallada acerca de la detención firme endoteliales barreras 22. Ensayos de transmigración como se demuestra aquí permiten el análisis de la transmigración de células inmune 21, 23, 24, 25, 26, 27, 28, 29. Uso de la humana en sangre modelo in vitro de barrera cerebro, recientemente hemos podido demostrar que un Migr mayorcapacidad Atory de CD56 CD16 brillante dim / - células NK en comparación con su CD56 dim CD16 + homólogos se reflejó por un predominio de este subconjunto de células NK en el compartimento intratecal 21. Por lo tanto, nuestra configuración experimental parece ser adecuado para imitar la situación 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

extravasación de linfocitos en el sistema nervioso central (SNC) es fundamental para la vigilancia inmune. alteraciones relacionadas con la enfermedad de extravasación de linfocitos podrían dar lugar a cambios fisiopatológicos en el SNC. Por lo tanto, la investigación de la migración de linfocitos en el SNC es importante entender las enfermedades inflamatorias del sistema nervioso central y para desarrollar nuevos enfoques de terapia. Aquí presentamos un modelo in vitro de la barrera sangre-cerebro humano para estudiar la extravasación de linfocitos. células endoteliales microvasculares del cerebro humano (HBMEC) se cultivan confluently en un tereftalato de polietileno poroso transwell insertar para imitar el endotelio de la barrera sangre-cerebro. La función de barrera es validado por zonula occludens inmunohistoquímica, la resistencia eléctrica transendotelial (TEER) mediciones así como el análisis de evans permeación azul. Este modelo permite la investigación de la diapedesis de los subgrupos de linfocitos raros tales como CD56 CD16 brillante dim / - las células NK. Ademmineral, los efectos de otras células, citocinas y quimiocinas, alteraciones relacionadas con la enfermedad, y regímenes de tratamiento distintos sobre la capacidad migratoria de los linfocitos puede ser estudiado. Por último, el impacto de los estímulos inflamatorios, así como diferentes regímenes de tratamiento en la barrera endotelial pueden ser analizados.

Se muestran los resultados representativos que muestran transmigración de las células NK y subconjuntos de células T utilizando el hematoencefálica modelo de barrera humana (Figura 1A). La integridad de la monocapa HBMEC fue validado por tinción de la molécula de unión estrecha mediciones de resistencia eléctrica transendotelial (TEER) ZO-1, y evans permeación azul (Figura 1B). Después de 3 - 4 días de cultivo HBMEC expresa la molécula de uni...

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.

Análisis de linfocitos extravasación El uso de una<em&gt; In Vitro</em&gt; Modelo de la barrera sangre-cerebro humano

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

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Immunology and Infection

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

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

Video: 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.

Aislamiento de linfocitos T humanos, cultura y análisis de la migración 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 ...

Investigación

Inmunología e Infección

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.

Punción en el dedo de sangre Metodología de muestreo para la determinación de la inducida por el ejercicio apoptosis de los linfocitos

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 Supresión como herramienta de cribado para el reconocimiento de un estado inicial de desequilibrio inmunológico

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.

Un<em&gt; In vitro</em&gt; Modelo para estudiar la heterogeneidad de diferenciación de los macrófagos humanos y polarización

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.

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.

La investigación de los efectos de los probióticos en antineumocócica Colonización El uso de un<em&gt; In Vitro</em&gt; Ensayo de adherencia

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.

Desarrollo de una<em&gt; In vitro</em&gt; Sistema modelo para estudiar la interacción de<em&gt; Equus</em&gt;<em&gt; Caballus</em&gt; IgE con su receptor de alta afinidad Fc 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 ...

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.

El aislamiento de linfocitos de la piel humana para el Análisis y fenotípica<em&gt; Ex Vivo</em&gt; Cultivo Celular

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.

Un<em&gt; In Vitro</em&gt; Modelo de la barrera sangre-cerebro mediante Espectroscopia de Impedancia: Un enfoque en la interacción de células T de la célula 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.

Dextrano mejora la eficiencia de transducción lentivirales de las células NK primaria murinas y humanas

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

Análisis de linfocitos extravasación El uso de una

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Aislamiento de linfocitos T humanos, cultura y análisis de la migración In Vitro

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Dextrano mejora la eficiencia de transducción lentivirales de las células NK primaria murinas y humanas