Hirschil Trust, Michael Saperstein Medical Scholars fondos de investigación, y la participación de la NYU Whitehead apoya este trabajo.

1. Revestimiento de la microplaca Wells Nota: El objetivo de este paso es a las superficies de poliestireno capa con la ICAM-1 para servir como ligandos para células T de LFA-1. <ol><li> Preparar las siguientes soluciones: <ol><li> Preparar la solución de recubrimiento mediante la resuspensión recombinante de ICAM-1 con solución salina tamponada con fosfato (PBS) (NaCl 137 mM, 2,7 mM de KCl, 10 mM de Na 2 HPO 4, 2 mM de KH 2 PO 4, pH 7,4) enriquecida con calcio (1 mM CaCl2) y magnesio (2 nM de MgCl 2). Asegúrese de que la concentración final de ICAM-1 es de 10 g / ml, elaborados a partir de una solución madre de 0,7 mg / ml y se mantuvieron en un -80 ° C congelador. </li><li> Preparar la solución de la adhesión mediante el enriquecimiento de PBS con 0,5% humana (o bovina) de albúmina de suero (HSA / BSA), 2 mM de MgCl 2, y 1 mM de CaCl2. </li><li> Prepare la solución de lavado añadiendo 2 mM MgCl 2 y 1 mM CaCl 2 a PBS. Calentar todas las soluciones a 37° C antes de su uso. </li></ol></li><li> Lávese 24 pozos con 50 l de solución de lavado. Esto incluirá pozos sin lavar (células tratadas con PMA) positivos, y (pozos sin revestimiento) de los controles negativos. Para cada condición establecida por lo menos tres pozos (por triplicado). </li><li> Añadir 50 l de solución de revestimiento a cada pocillo. No agregue a los pocillos de control sin revestir. Incubar durante una hora en el interior 37 ° C incubadora. </li><li> Aspirar la solución de recubrimiento suavemente, sin permitir que la punta toque el fondo de los pocillos. Lavar una vez con 50 l de solución de lavado. </li><li> Añadir 50 l de solución de adherencia y se incuba la microplaca durante una hora en el interior 37 ° C incubadora. </li><li> Aspirar con cuidado y lavar una vez con 50 l de solución de lavado. Utilice la placa en el mismo día, sin embargo es posible que la mantenga a 4 ° C durante la noche, y usarlo al día siguiente. </li></ol> 2. T aislamiento de células de la sangre <ol><li> Añadir humana T cóctel de enriquecimiento de células a 50 l / ml de of sangre entera (por ejemplo, para 3 ml de anticoagulante (heparina, EDTA o citrato) de sangre entera, añadir 150 l de cóctel). Mezclar bien. </li><li> Incubar 20 min a temperatura ambiente. </li><li> Para un tubo de centrífuga de 50 ml añadir 3 ml de sangre tratada y un volumen igual de PBS (sin calcio y magnesio) enriquecida con 1% de D-glucosa a temperatura ambiente. Mezclar suavemente con una pipeta Pasteur. </li><li> Añadir 15 ml de Ficoll-Paque PLUS al tubo de centrífuga. </li><li> Capa con cuidado los 6 ml diluidos muestra de sangre en el medio de aislamiento de linfocitos. </li><li> Se centrifuga a 400 × g durante 20 min a 20 ° C con rotura de apagado. </li><li> Trasvasar la capa superior con una pipeta Pasteur limpia, dejando la capa de linfocitos imperturbable en la interfase. La capa superior de plasma se puede guardar para su uso posterior. Con una pipeta Pasteur limpia transferir la capa de linfocitos a un tubo de centrífuga limpio. </li><li> Añadir 3 volúmenes (9 ml) de PBS para los linfocitos. Suspender las células suavemente por la Dra.ala ellos dentro y fuera de una pipeta Pasteur. </li><li> Se centrifuga a 400 × g durante 5 min a 20 ° C. </li><li> Eliminar el sobrenadante. Los linfocitos ahora deben ser suspendidos en el medio apropiado para la aplicación. Transferir las células a un frasco de cultivo T-75 en 20 ml de RPMI 1640 que contenía 10% de FBS, 1% de penicilina / estreptomicina. </li></ol> 3. Preparación de las células Nota: Un requisito previo es que las células se pueden etiquetar con un reactivo fluorescente. En este protocolo utilizar carboxi éster de succinimidilo de fluoresceína (CFSE), proteína fluorescente sin embargo verde (GFP) son células que expresan una alternativa aceptable. <ol><li> Cuente 2,4 x 10 6 células T (1 x 10 5 para cada pocillo) usando un hemocitómetro. </li><li> Resuspender las células en medios de cultivo que carece de suero (privación de suero). Se incuban las células durante 2 horas en la incubadora a 37 ° C. </li><li> Centrifugar las células durante 5 min (400 xg). Aspirar los medios de comunicación yresuspender en 1 ml de PBS. </li><li> Añadir CFSE a 1:1000 dilución (caldo hecho con 5 mM). Cubra la luz y se incuba durante 8 minutos a temperatura ambiente. </li><li> Se detiene la reacción mediante la adición de 10 ml de 37 ° C PBS y centrifugar a 400 xg durante 5 min. </li><li> Vuelva a suspender el sedimento celular con 1,2 ml de solución de adherencia precalentado (1 x 10 5 células por 50 l). </li></ol> 4. Estimulación de las células Nota: Con el fin de iniciar la adhesión, las células deben ser estimulados. En el siguiente experimento, las células son estimuladas a través del TCR usando anticuerpos anti-CD3 de reticulación, sin embargo solubles SDF-1 podría servir como una alternativa. Dependiendo del objetivo de los experimentos, varios reactivos farmacológicos podrían añadirse antes o durante la estimulación para estudiar el impacto de la adhesión celular. Forbol miristato acetato (PMA) es un éster de forbol que es estructuralmente similar a la segunda diacil-glicerol mensajero (DAG) y por lo tanto activa múltiples quinasas en sentido descendente del TCR (principalmente la proteína quinasa C); Aquí se usa como un control positivo. <ol><li> Calentar la microplaca a 37 ° C. </li><li> Dividir las células en 8 vacíos tubos de 1,5 ml (150 mu l en cada tubo), un tubo para cada condición. </li><li> Tratar a las células en consecuencia: <ol><li> Estimular un tubo con PMA a 10 ng / ml para servir como control positivo. Mantenga tres tubos sin tratar, para servir como control para la estimulación (&quot;sin estimulación&quot;), el control de la carga sin lavar (&quot;sin lavado&quot;), y el control de ICAM-1 capa (&quot;sin recubrimiento&quot;). </li><li> Estimular cuatro tubos con diferentes concentraciones de anticuerpos anti-CD3 (0,1, 1, 5, y 10 mg / ml). Continúe en el siguiente paso sin retrasos. </li></ol></li><li> Alícuota de 50 l de mezcla de células estimuladas en cada pozo vacío. El número final de células por pocillo es 1 x 10 5. </li><li> Coloque la microplaca en el 37 ° C incubadora durante 15 min. Nota: Algunas líneas de células T requieren estimulación más corto<html> tiempo ción. Durante este tiempo, las células se establecerán a la parte inferior de los pocillos y se adhieren a los ligandos diana. </li></ol> 5. Lavamiento células no adherentes Nota: El objetivo de este paso es eliminar las células que no eran capaces de formar contactos estrechos con las superficies recubiertas con ligando. Utilizar una pipeta multicanal para este paso con el fin de garantizar que las mismas fuerzas físicas se aplican a todos los pocillos. Es importante mantener los pocillos de control indicados sin lavar para ayudar en el cálculo del porcentaje de células adherentes. <ol><li> Añadir 150 l de solución de adherencia caliente para cada uno pozos. Agitar suavemente la placa durante unos segundos antes de retirar el medio de los pocillos. No toque el fondo de los pozos con las puntas. </li><li> Repita este paso tres veces. Cambie la dirección de la pipeta al pipetear el buffer de entrada y salida. </li></ol> 6. La determinación del porcentaje de células adherentes _content &quot;&gt; Nota: En este paso un lector de placas fluorescente se utiliza para medir la intensidad de fluorescencia dentro de cada pocillo La intensidad está en correlación directa con el número de las células adherentes.. <ol><li> Encienda el lector de placas. Abra el software de lector de placas haciendo clic en el icono de acceso directo en el escritorio. <ol><li> En el menú &quot;Tareas&quot;, haga clic en &quot;Nuevo&quot; para crear un nuevo protocolo. </li><li> En el menú &quot;Actions&quot; elija la opción &quot;Leer&quot;. Haga clic en &quot;intensidad fluorescente&quot; y pulsa la pestaña &quot;Ok&quot;. </li><li> Desde el menú desplegable, seleccione &quot;longitud de onda de excitación 485&quot; y &quot;longitud de onda de emisión 528&quot;. Pulse la pestaña &quot;Ok&quot;. </li><li> En el menú de opciones &quot;óptica&quot;, seleccione &quot;Bottom&quot; y pulsa la pestaña &quot;Ok&quot;. Volver al menú &quot;Actions&quot; y ajustar la temperatura a 37 ° C y haga clic en &quot;Ok&quot;. </li><li> Inserte la microplaca en el lector de placas yhaga clic en &quot;Ejecutar&quot;. El resultado se puede exportar a hoja de cálculo Excel. </li></ol></li><li> Calcular el porcentaje de células adherentes utilizando la siguiente fórmula: Porcentaje de células adherentes = (intensidad de fluorescencia media leer en los pocillos lavados) / (intensidad de fluorescencia media de leer en las células sin lavar) x 100%. </li></ol>

<ol>
	<li>Dustin, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membranes+as+messengers+in+T+cell+adhesion+signaling.">Membranes as messengers in T cell adhesion signaling.</a> Nat Immunol. 5 (4), 363-372 (2004).</li><li>Mor, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTPases+and+LFA-1+reciprocally+modulate+adhesion+and+signaling.">GTPases and LFA-1 reciprocally modulate adhesion and signaling.</a> Immunol Rev. 218, 114-125 (2007).</li><li>Rose, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+modulation+and+signaling+in+leukocyte+adhesion+and+migration.">Integrin modulation and signaling in leukocyte adhesion and migration.</a> Immunol Rev. 218, 126-134 (2007).</li><li>Alon, R., Feigelson, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine-triggered+leukocyte+arrest:+force-regulated+bi-directional+integrin+activation+in+quantal+adhesive+contacts.">Chemokine-triggered leukocyte arrest: force-regulated bi-directional integrin activation in quantal adhesive contacts.</a> Curr Opin Cell Biol. 24 (5), 670-676 (2012).</li><li>Griffith, J. W., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+cells+in+motion:+migrating+toward+improved+therapies.">Targeting cells in motion: migrating toward improved therapies.</a> Eur J Immunol. 43 (6), 1430-1435 (2013).</li><li>Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+molecules+and+actin+cytoskeleton+at+immune+synapses+and+kinapses.">Cell adhesion molecules and actin cytoskeleton at immune synapses and kinapses.</a> Curr Opin Cell Biol. 19 (5), 529-533 (2007).</li><li>Springer, T. A., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+inside-out+signaling+and+the+immunological+synapse.">Integrin inside-out signaling and the immunological synapse.</a> Curr Opin Cell Biol. 24 (1), 107-115 (2012).</li><li>Mor, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=D1+regulates+lymphocyte+adhesion+via+upregulation+of+Rap1+at+the+plasma+membrane.">D1 regulates lymphocyte adhesion via upregulation of Rap1 at the plasma membrane.</a> Mol Cell Biol. 29 (12), 3297-3306 (2009).</li><li>Bivona, T. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rap1+up-regulation+and+activation+on+plasma+membrane+regulates+T+cell+adhesion.">Rap1 up-regulation and activation on plasma membrane regulates T cell adhesion.</a> J Cell Biol. 164 (3), 461-470 (2004).</li><li>Zeltzer, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diminished+adhesion+of+CD4++T+cells+from+dialysis+patients+to+extracellular+matrix+and+its+components+fibronectin+and+laminin.">Diminished adhesion of CD4+ T cells from dialysis patients to extracellular matrix and its components fibronectin and laminin.</a> Nephrol Dial Transplant. 12 (12), 2618-2622 (1997).</li><li>Bhattacharyya, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Both+adhesion+to+immobilized+vitronectin+and+Fc+epsilon+RI+cross-linking+cause+enhanced+focal+adhesion+kinase+phosphorylation+in+murine+mast+cells.">Both adhesion to immobilized vitronectin and Fc epsilon RI cross-linking cause enhanced focal adhesion kinase phosphorylation in murine mast cells.</a> Immunology. 98 (3), 357-362 (1999).</li><li>Dustin, M. L., Groves, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+signaling+clusters+in+the+immune+synapse.">Receptor signaling clusters in the immune synapse.</a> Annu Rev Biophys. 41, 543-556 (2012).</li></ol>

Los autores no tienen nada que revelar.

Hay varios ensayos para estudiar las señales a la LFA-1 de activación y adhesión de células T 12. La citometría de flujo se utiliza para medir la LFA-1 estados de afinidad en las células vivas utilizando anticuerpos monoclonales que se unen selectivamente a cualquiera de doblado o extendido de LFA-1. Una de las limitaciones de este método es que no tiene en cuenta la avidez de las integrinas. Migración ensayos son herramientas útiles, pero miden la migración, y no la adhesión miserable en un punto ...

Adhesión de los linfocitos T es un proceso fundamental en la respuesta inmune 1. Se requiere para la interacción de células T con células endoteliales decoración de las paredes de los capilares, para el antígeno de células presentadoras de barrido (APC) dentro de los ganglios linfáticos, y para la formación de sinapsis inmunológica (IS) con las células diana 2. Estos requisitos son funcionalmente y cinéticamente distintas. El proceso de la extravasación de linfocitos consiste quimioatracción, laminados, la adhesión firme y la transmigración. La transición de la rueda a la adhesión firme requiere que las células T para responder a una señal de receptor acoplado a proteína G rápidamente. Esta respuesta se obtiene una interacción ligando integrina que ralentiza y detiene la célula de rodaje 3. Un cambio inmediato en la avidez de la integrina medie este proceso. La migración requiere interacciones betweenTcells dinámicas y las células endoteliales con la formación de adherencias en el &#39;front-end&#39; y la rotura de adherencias en la &quot;parte trasera&quot;con un intervalo de aproximadamente un minuto entre la formación y la ruptura 4. El IS forma inminutes, pero debe permanecer intacto durante horas 6. Curiosamente, una molécula de adhesión, la función de los linfocitos miembro de la familia de integrinas antígeno asociado (LFA) - 1, es esencial para todos estos procesos 5. LFA-1 media la adhesión a través de interacciones con varios miembros de la superfamilia de las inmunoglobulinas. El ligando más ampliamente estudiado con la mayor afinidad a la LFA-1 es la molécula de adhesión intercelular (ICAM) -1. Linfocitos circulantes no activadas expresan baja afinidad de LFA-1 en la superficie celular, y por lo tanto son incapaces de adherirse a la ICAM-1 recubiertos superficies. LFA-1 afinidad es variable y regulada por varios eventos de señalización tales como la activación de la proteína G de receptores acoplados, la estimulación de citoquinas, y las señales mediadas por receptores de células T (TCR). La forma de alta afinidad resultante de la LFA-1 transmite la activación intracelular al espacio extracelular tediante interacciones con la ICAM-1. Esta vía se denomina dentro-fuera de señalización 7. Del mismo modo, la señalización a través de LFA-1 desde el espacio extracelular se llama fuera-en la señalización. La cascadas de señalización intracelular que participan en el interior-y exterior-en la señalización son un foco importante de la investigación actual. La pequeña GTPasa Rap1 ha surgido recientemente como el componente clave de dentro a fuera de señalización que es común tanto a la ligadura de TCR y la señalización de citoquinas 8. El papel crítico de Rap1 en activación de la integrina se pone de relieve por el descubrimiento de que la sobreexpresión de Rap1 estimula la adhesión dependiente de integrina de las células T, mientras que la adhesión de células T está bloqueada por la expresión de dominante negativo Rap1 9. Estos avances en nuestra comprensión de la regulación de la integrina por Rap1 han llevado a cabo utilizando herramientas vitro. Entre ellos se encuentra el ensayo de adhesión estática se describe aquí. El objetivo general de este método es el estudio de Tadhesividad de células a la ICAM-1 las superficies recubiertas. Más específicamente, se utiliza para medir y cuantificar objetivamente la LFA-1 afinidad hacia sus ligandos contador en células vivas en tiempo real, bajo diferentes condiciones. Esta técnica utiliza pocillos de poliestireno recubiertas con ICAM-1 para imitar las superficies celulares que las células T interaccionan con. Muchos ensayos de adhesión de células T estáticos descritos anteriormente fueron experimentalmente complejo. Estos ensayos a menudo necesarios para las células T que se marcaron radiactivamente, utilizan células corneales bovinas cultivadas para crear una matriz extracelular como el sustrato para la adhesión de las células T, o llamados para no fisiológica estimulación de células T durante una duración prolongada para promover la adhesión de células T 10. El uso de la medición fluorométrica para cuantificar las células T después de la incubación de adherencia es un método más sensible y precisa de la cuantificación en comparación con la citometría de flujo y microscopía, como se utilizan en muchos otros sistemas de ensayo 11. Además, solo microscópicas de célulasIC análisis de la localización de la integrina no permite el análisis amplio, la población basada en la misma forma que la medición fluorométrica. Mientras que la activación de LFA-1 anticuerpos específicos de cada estado están disponibles comercialmente, estos anticuerpos ofrecen baja sensibilidad en relación con el método descrito aquí. La principal ventaja sobre las técnicas alternativas es su simplicidad y la capacidad de examinar múltiples condiciones experimentales simultáneamente. Al considerar este método para una aplicación específica, se debe tener en cuenta que las células T deben ser seleccionados negativamente, recién aisladas, y se etiquetan con un marcador fluorescente.

<table border="0" cellpadding="0" cellspacing="0" width="828">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Plate reader</td>
			<td style="width:160px;">BioTek&#160;</td>
			<td style="width:136px;">Synergy H1 Hybrid</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Multichannel pipette</td>
			<td style="width:160px;">Fisher</td>
			<td style="width:136px;">21-377-829</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">96-well microplate with optical bottom</td>
			<td style="width:160px;">Corning Costar</td>
			<td style="width:136px;">3603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Recombinant ICAM-1</td>
			<td style="width:160px;">R&#38;D Systems</td>
			<td style="width:136px;">ADP4-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Anti-CD3 antibodies</td>
			<td style="width:160px;">Ancell</td>
			<td style="width:136px;">144-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">PMA</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:136px;">79346</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">BSA</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:136px;">A2058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">CFSE</td>
			<td style="width:160px;">Molecular Probes</td>
			<td style="width:136px;">C1157</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">RPMI</td>
			<td style="width:160px;">Gibco</td>
			<td style="width:136px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">DPBS containing calcium and magnesium</td>
			<td style="width:160px;">Gibco</td>
			<td style="width:136px;">14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Peripheral lymphocytes</td>
			<td style="width:160px;">e.g. mouse or human</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>499609</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Open Gen 5 software</td>
			<td>BioTek</td>
			<td>Version 2.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ficoll-Paque PLUS</td>
			<td>GE Healthcare</td>
			<td>71-7167-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 and 50 mL centrifuge tubes</td>
			<td>Fisher</td>
			<td>352099, 352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable plastic Pasteur pipettes</td>
			<td>Fisher</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T-75 culture flasks</td>
			<td>Fisher</td>
			<td>50-754-1366</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RosetteSep T cell enricment kit</td>
			<td>StemCell Technologies</td>
			<td>15061</td>
			<td></td>
		</tr>
	</tbody>
</table>

static adhesion assay for the study of integrin activation in t lymphocytes

Se requiere la adhesión de linfocitos T para múltiples funciones de las células T, incluyendo la migración a los sitios de inflamación y formación de sinapsis inmunológica con células presentadoras de antígeno. Células T lograr la adhesión regulada mediante el control de las propiedades adhesivas de las integrinas, una clase de moléculas de adhesión celular que constan de pares de proteínas transmembrana heterodiméricas que interactúan con moléculas diana en las células asociadas o matriz extracelular. El más prominente de la integrina células T es función de los linfocitos antígeno asociado (LFA) -1, compuesto por subunidades αL y β2, cuyo objetivo es la molécula de adhesión intracelular (ICAM) -1. La capacidad de una célula T para controlar la adherencia se deriva de la capacidad de regular los estados de afinidad de las integrinas individuales. Dentro de señalización de salida describe el proceso por el cual las señales dentro de una célula causan los dominios externos de las integrinas para asumir un estado activado. Mucho de nuestro conocimiento de estos complejos fenómenos se basa en mecanicistaLos estudios realizados en simplificación en sistemas de modelos in vitro. El ensayo de adhesión de linfocitos T se describe aquí es una excelente herramienta que permite a las células T para que se adhieran a las moléculas diana, en condiciones estáticas, y luego utiliza un lector de placas de fluorescencia para cuantificar la adhesividad. Este ensayo ha sido útil en la definición de sustancias-estimulador o inhibidor de adhesión que actúan sobre los linfocitos, así como la caracterización de los eventos de señalización implicadas. Aunque se ha descrito aquí para la LFA-1 - ICAM-1 de adhesión mediada; este ensayo puede ser fácilmente adaptado para permitir el estudio de otras interacciones adhesivas (por ejemplo, VLA-4 - fibronectina).

A continuación se muestra un ejemplo de ensayo de adhesión usando células T primarias estimuladas con diversas concentraciones de anticuerpos anti-CD3. Es útil conocer la fluorescencia de las células sin lavar (es decir, la carga total). Células no estimuladas sirven como un control negativo y las células tratadas de PMA son el control positivo para anticuerpos anti-CD3. Las células sembradas en pocillos no recubiertos sirven como un control para la ICAM-1. En un experimento típico, el porcentaje de c?...

Watch this Scientific Journal Video about Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes at JoVE.com

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

Ensayo de adhesión estática es una herramienta poderosa que se puede utilizar para modelar las interacciones entre los linfocitos T y otros tipos de células. Interacciones se generan mediante la inyección de células T marcadas en pocillos recubiertos con moléculas de adhesión, mientras que un lector de placas se utiliza para cuantificar el número de células adherentes después de lavados de serie.

Ensayo de adherencia estática para el Estudio de la integrina activación en linfocitos T

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

static-adhesion-assay-for-study-integrin-activation-t

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Cell Biology

Immunology and Infection

Unit 1

Cell-Matrix Interactions

SCIENTISTS IN ACTION

15.9K vistas.  New York University School of Medicine.  Ensayo de adhesión estática es una herramienta poderosa que se puede utilizar para modelar las interacciones entre los linfocitos T y otros tipos de células. Interacciones se generan mediante la inyección de células T marcadas en pocillos recubiertos con moléculas de adhesión, mientras que un lector de placas se utiliza para cuantificar el número de células adherentes después de lavados de serie. 

Video: Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Métodos para evaluar la Beta mediada por la muerte celular por linfocitos T citotóxicos

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Investigación

Inmunología e Infección

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

Ensayo in vitro de la adhesión bacteriana a las células epiteliales de mamíferos

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

La introducción de la tensión de cizallamiento en el Estudio de la adhesión bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Diferenciación dirigida de células madre pluripotentes inducidas hacia los linfocitos T

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

Cuantitativo<em&gt; In vitro</em&gt; Ensayo para medir la adhesión de neutrófilos al Activado Primaria células endoteliales microvasculares humanas en condiciones estáticas

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Aislamiento y diferenciación de Th17 Naïve linfocitos T CD4

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Un ensayo de adhesión de flujo para estudiar Leucocyte Reclutamiento para hepático humano sinusoidales endotelio en condiciones de estrés de cizalla

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Ensayo de adhesión de neutrófilos Cámara de flujo humano

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In vitro e in vivo modelo para estudiar la adhesión bacteriana a la pared del recipiente en condiciones de flujo

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Protocolo Aislamiento eficiente para B y linfocitos T a partir Humanos Palatine amígdalas

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...