La financiación de este proyecto fue proporcionado por un premio de la Sociedad Americana del Cáncer Research Scholar, (06-024-01-CSM de Carlos J. Dimitrov), los Institutos Nacionales de la Salud / Beca Nacional Cancer Institute (RO1CA118124 a Charles J. Dimitrov), y de los Institutos Nacionales de Salud / Instituto Nacional de Artritis y Enfermedades Musculoesqueléticas y de la Piel Enfermedades de subvención (P30 AR042689 al Dr. Thomas S. Kupper, el Hospital Brigham and Women, en Boston, MA).

1. Preparación de hBMEC (HBMEC-60 Cell) monocapa en placas de Petri para la cámara de flujo <ol><li> Escudo de 35 x 10 mm cultivo de tejidos platos con 2 ml de fibronectina 20μg/ml (en PBS) durante la noche a 4 ° C o durante 3 horas a 37 º C. </li><li> Aspirar la solución de fibronectina de los platos y añadir 1,5 x10 5 células HBMEC-60 [Media 199 con HEPES y glutamina, un 10% de SFB, 10% de suero humano, 5 unidades / ml de heparina, 1ng/ml recombinante factor de crecimiento de fibroblastos, 1% de penicilina- estreptomicina] para el plato y les permiten crecer a la confluencia&gt; 90%. (Esto toma 2 días) </li><li> Aspirar el medio de crecimiento y, en un máximo de regular la expresión de E-selectina, añadir un nuevo medio con IL-1β 50ng/mL de 4-6 horas. Monocapas de células ya están listos para rodar ensayo. Para bloquear la función E-selectina, neutralizantes anti-humana de E-selectina Moab (clon BBIG-E4 (5D11)) se pueden agregar a 20μg/ml de ~ 1 hora a 37 ° C. </li></ol> 2. Preparación de las células progenitoras hematopoyéticas KG1a de la cámara de flujo <ol><li> Células hematopoyéticas humanas KG1a progenitoras se cultivan hasta la confluencia (1x10 6 células / ml) en RPMI-1640 con glutamina y el 10% de SFB / 1% penicilina-estreptomicina y se pipetean directamente desde el frasco para su uso en el ensayo de cámara de flujo. </li><li> El número de células se calcula utilizando un hemocitómetro y luego las células se resuspendieron a 1 x 10 6 células / ml en 10 mM HEPES con HBSS (H / H Buffer) y 2 mM de CaCl2 </li><li> Las células se guardan en hielo hasta su uso en el ensayo. </li></ol> 3. Preparación de microscopio, cámara de flujo placas paralelas y bomba de jeringa (Figura 1) <img src="/files/ftp_upload/old/1009_2009_1_26_7/1009_figure1.png" alt="Figura 1" width="600" height="409" /> Figura 1. Ilustración de Análisis de placas paralelas de flujo de la Cámara. Microscopio invertido, bomba de Harvard jeringa, equipo de cámara / y de placas paralelas cámara de flujo se colocan en una disposición óptima para el análisis celular eficiente y reproducible. <ol><li> A su vez en el poder y fuente de luz para microscopio invertido y el poder de la bomba de jeringa y seleccionar el objetivo de 10x en el microscopio. </li><li> Coloque 50 ml tubo cónico en el soporte y se llenan de H / H y 2 mM de CaCl2. </li><li> Colocar 5 ml tubos de plástico en el soporte de tubos de ensayo por debajo del tubo cónico de 50 ml </li><li> Coloque 60 ml jeringa en la bomba de jeringa (Asegúrese de que tanto el émbolo de la jeringa y el cuerpo son seguros). </li><li> Adjuntar 3-way llave de paso para poner fin a 60 ml de la jeringa y coloque la jeringa de 5 ml de 3-way llave de paso. </li><li> Lugar de flujo paralelo aparato Cámara (GlycoTech, Inc.) en platina del microscopio con el tubo de entrada a la derecha, el tubo de salida a la izquierda y tubo de vacío a la espalda. </li><li> Conecte el tubo de vacío a vacío y válvula de aire libre para permitir que un aparato de placas paralelas a adherirse a la etapa. (Esta es la prueba si la falda de vacío en la cámara de flujo es hermético.) Cierre la válvula de aire. </li><li> Conecte la línea de salida de la tubería (a la izquierda del microscopio) para 3-way llave de paso. Aparato está preparado para la puesta en plancha monocapa HBMEC-60. </li><li> Lugar HBMEC-60 monocapa placa y el lugar en el centro del microscopio. </li><li> Activar el vacío y la posición del aparato de la cámara de flujo por encima de la placa de tal manera que la plancha monocapa está en el centro. </li><li> Baje suavemente el aparato de la cámara de flujo sobre la placa de monocapa HBMEC-60 y los aparatos que el flujo de la cámara de succión hacia abajo. (No debe haber ningún ruido de escape de aire y en este momento puede ser necesario aplicar una presión a la cámara para comprimir la junta de goma.) </li><li> Una vez vacío, se ha establecido, tubo lugar la entrada (a la derecha) en el tubo de 50 ml cónicos que contengan H / H con 2 mM de CaCl2. </li><li> Abierto de 3 vías llave de paso para permitir el flujo dentro de la jeringa 60 ml y la bomba a cabo en el &quot;modo de la bomba.&quot; </li><li> Para eliminar el aire de las líneas de entrada / salida sin necesidad de levantar las células en la placa, use la bomba de jeringa fijada en 2mL/minute para llenar el tubo de admisión, entonces rápidamente se puso a 0,5 ml / minuto justo antes de que el fluido llega al aparato. Es imperativo que este primer set con cuidado y evitar burbujas de aire que se levante la monocapa de la placa. </li><li> Una vez que el líquido se ve que se mueven dentro de la jeringa de 60 ml, la cámara de flujo y bomba de jeringa se han preparado y la cámara de flujo está listo para su uso. Esta disposición se repetirá para cada corrida de entrada de la célula y en cada nueva placa es necesario. </li></ol> 4. La captura de los leucocitos Eventos Rolling utilizando elementos NIS <ol><li> Abrir NIS elementos en el escritorio. </li><li> Pulse el botón &quot;play&quot; icono de la imagen en vivo. Exposición automática le permite ver la placa más fácil por el momento y es importante para el enfoque. </li><li> Asegúrese de que microscopio se centra correctamente en la monocapa celular. </li><li> Una vez centrado, vuelva a ajustar la exposición a una luz de marco / segundo y el ajuste en el microscopio. La imagen deberá aparecer en el ordenador y ya no será visible en el visor microscopio debido a la poca luz. Histograma de la luz puede ser adajustado para eliminar la exposición de fondo o mejorar la claridad de la célula. </li><li> Haga clic en Bin Bin 2X2 o en vivo para obtener una imagen que es ideal para la captura de películas </li><li> Haga clic en &quot;Macro&quot; en la barra de menú y selecciona &quot;escribir en el puerto&quot;, seleccionar el puerto &quot;COM3&quot; y &quot;abierto&quot;. (Se abre el puerto de coordinar con la bomba de jeringa. </li><li> Pipeta de 1 ml de suspensión celular KG1a en el tubo de ensayo de 5 ml cerca de la línea de tubos de entrada y luego colocar la tubería de entrada de línea en la parte inferior del tubo de ensayo. </li><li> Ir a la barra de menú, haga clic en &quot;Capture&quot;, &quot;lapso de tiempo de adquisición.&quot; </li><li> Un nuevo menú pop-up. Seleccione el protocolo de 210 segundos que dura, esta es la duración del programa para el despliegue de HBMEC-60 células. (Cada fase está programada para tomar una foto cada segundo durante la duración del protocolo de la bomba. También es configurado para enviar un comando a través del puerto COM3 para arrancar la bomba.) Si lapso de tiempo se interrumpe, la bomba no se detiene por su propia cuenta y se debe restablecer. Pulse Cancel para volver a vivir de la imagen. </li><li> Establecer la bomba de jeringa a &quot;modo de programación&quot;, según el manual de instrucciones. (El modo de programación permite la programación manual de las tasas de flujo específico (es decir, los niveles de tensión de corte) que se imparte dentro de la cámara y dictar las interacciones KG1a de células durante el monocapa de células HBMEC-60.) Configuración de la celda KG1a rodando por las células hBMEC fueron los siguientes 4,2 dinas / cm 2 durante 45 s, 0,3 dinas / cm 2 durante 60 s, y el aumento progresivo cada 15 s hasta un máximo de 4,2 dinas / cm 2 </li><li> La tensión de cizallamiento (W º) en la cámara se calculará de acuerdo con la siguiente ecuación: W = 6μQ st / a b 2, donde μ es la viscosidad del estimado de los medios de comunicación (0,0076 P), Q es el caudal en ml / s, a es la altura del canal (espesor de la junta) en cm (0,03 cm), y b es el ancho del canal en cm (0,5 cm). Los caudales se regula con el modo de programa de la bomba de jeringa. </li><li> Microscopio y la computadora está listo para la captura de fotografías a intervalos. Pulse el botón &quot;Inicio, Ejecutar&quot; en el equipo para comenzar a adquirir los datos. Mientras se ejecuta el programa, asegúrese de agregar los medios de comunicación para el tubo de ensayo que contienen las células KG1a con el fin de mantener el tubo de entrada / salida completa del medio (Asegúrese de que no hay burbujas de aire). </li></ol> 5. La dependencia de sialilación Terminal y glicoproteína de superficie de E-selectina mediada por células KG1a 5. Rolling (Figura 2, Videos Clips de 5.1 a 5.5) <ol><li> Dosificación de los celulares KG1a rodando por no estimulados HBMEC-60 células. (Control negativo) las células KG1a se prepararon y se infunde en la cámara de flujo en vivo no IL-1β estimulada HBMEC-60 como se describió anteriormente. </li><li> Dosificación de los celulares KG1a rodando por HBMEC-60 células pre-tratadas con IL-1β. (E-selectina, la dependencia de control de) las células KG1a se prepararon y se infunde en la cámara de flujo en vivo de IL-1β estimulada HBMEC-60 como se describió anteriormente. </li><li> Dosificación de los celulares KG1a rodando por HBMEC-60 células pre-tratadas con IL-1β y luego neutralizar anti-humano de E-selectina Moab. (E-selectina, la dependencia de control) las células KG1a se prepararon y se infunde en la cámara de flujo en vivo de IL- 1β estimulada HBMEC-60 previamente con anticuerpos neutralizantes humanos E-selectina moAb como se describió anteriormente. </li><li> Ensayo de la sialidasa digerir células KG1a rodando por IL-1β estimulada HBMEC-60. KG1a células pretratadas con 0.1U/ml Vibrio Cholerae neuraminidasa durante 1 hora a 37 ° C se prepararon y se infunde en la cámara de flujo en vivo de IL-1β estimulada HBMEC-60 como se describió anteriormente. </li><li> Ensayo de la proteasa-digerido celular KG1a rodando por IL-1β estimulada HBMEC-60. KG1a células pretratadas con la proteasa, la bromelina 0.2U/ml durante 1 hora a 37 ° C, se prepararon y se infunde en la cámara de flujo en vivo de IL-1β estimulada HBMEC-60 como se describió anteriormente. </li></ol> 6. El análisis de NIS-Elements-Capturado células KG1a y graficación de datos celular KG1a Rolling <ol><li> Después de la secuencia de tiempo se adquiere, guarde los datos. </li><li> Ir a &quot;medida&quot; pestaña en la barra de menú y selecciona &quot;definir el umbral.&quot; Mueve las barras para que las células deseadas son de color rojo. Seleccionar &quot;Todo&quot; y luego &quot;Aceptar&quot;. Todo el lapso de tiempo la secuencia debería ser ahora modificado con visibles las células rojas de rodadura. </li><li> Ir a &quot;medida&quot; y seleccione &quot;restricciones&quot;. (Las restricciones permiten al usuario excluir los objetos un umbral que no representan las células de laminación. Esto se debe principalmente el fondo creado por la monocapa HBMEC-60). Seleccione &quot;zona&quot; y un mínimo de entrada y los valores máximos para el área de las células de laminación. Repita este paso para &quot;elongación&quot; y &quot;circularidad&quot;. </li><li> Seleccione &quot;medida&quot; y seleccione &quot;crear binarios con restricciones&quot;, y establecer los valores de restricción para optimizar el NIS-Elements software de reconocimiento de las células de rodar en monocapa de células HBMEC-60. </li><li> Volver a &quot;medida&quot;, seleccione &quot;Los datos de campo&quot;, y luego seleccione &quot;datos reset&quot;. Cerrar este menú. </li><li> Volver a la &quot;medida&quot; y síSelect &quot;campo de lectura&quot;. </li><li> Abierta &quot;Medida&quot;, &quot;Los datos de campo&quot;, seleccione &quot;número u objetos&quot; y luego seleccionar &quot;todos los campos&quot; y exportar datos a Microsoft Excel. </li><li> Seleccione &quot;datos claros&quot; para preparar NIS-Elements adquisición de celulares para la recolección de datos siguiente. </li></ol> Resultados: <img src="/files/ftp_upload/old/1009_2009_1_26_8/1009_figure3.png" alt="Figura 3" width="600" height="382" /> Figura 3. Análisis de placas paralelas cámara de flujo de E-selectina actividad de unión en las células KG1a. Las células tratadas con sialidasa KG1a o de la proteasa fueron analizadas para rodar la eficiencia en monocapas de HBMEC-60 células pre-tratadas con IL-1β. Control negativo rodar experimentos se llevaron a cabo también mediante el análisis de células KG1a rodando por no IL-1β estimulada HBMEC -60 células o la IL-1β estimulada HBMEC-60 células tratadas con anticuerpos neutralizantes humanos E-selectina Moab (5D11). Las frecuencias de celular móvil se realizaron por triplicado y se analizaron con NIS-Elements. (* Significación estadística, p &lt;0,001) En este informe, basado en video, que proporciona una representación visual de cómo analizar los leucocitos - las interacciones de las células endoteliales en condiciones fisiológicas esfuerzo cortante. En particular, se analizó el papel de la E-selectina - E-selectina ligandos en la mediación de la inmovilización de leucocitos y laminados, un comportamiento adhesivo necesario para el compromiso de las actividades secundarias de unión más estable a través de receptores, como las integrinas y los receptores de ácido hialurónico. Usando la cámara de flujo de placas paralelas adaptado para su uso con un microscopio invertido, la bomba de Harvard jeringa, una cámara de vídeo y el ordenador / celular de adquisición de hardware, se realizaron experimentos en los que la E-selectina ligando + células KG1a se utilizaron como modelo de leucocitos y las células hBMEC (HBMEC- 60) estimulados con citoquinas pro-inflamatorias, IL-1β, fueron utilizados como la E-selectina + modelo humano de las células endoteliales (1-6). Como se informó anteriormente, se observó robusta célula KG1a rodando por IL-1β estimulada HBMEC-60 células en un rango de niveles de esfuerzo cortante en forma de E-selectina-dependiente (Figura 3) (diferencia estadísticamente significativa en comparación con las células KG1a rodando por no IL-1β HBMEC-60 estimula las células). Los controles negativos, que consistía en células KG1a rodando por no IL-1β estimulada HBMEC-60 o las células de la IL-1β estimulada HBEMC-60 células pretratadas con anti-humano de E-selectina mAb, se realizaron en paralelo para demostrar la dependencia de la estimulación de citoquinas y la expresión de E-selectina de la actividad de rodadura (Figura 3). Además, el análisis de rodadura de las células pretratadas con KG1a Vibrio cholerae neuraminidasa (sialidasa) o con la bromelina, una proteasa no específica conocida para la digestión de la E-selectina ligando glicoproteína de superficie, destacó la importancia de los residuos terminales de ácido siálico y la glicoproteína de superficie para la E-selectina ligando actividad (2,3).

<ol>
	<li>Dimitroff, C. J., Kupper, T. S., Sackstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+leukocytic+migration+to+inflamed+skin+with+a+novel+fluorosugar+modifier+of+cutaneous+lymphocyte-associated+antigen.">Prevention of leukocytic migration to inflamed skin with a novel fluorosugar modifier of cutaneous lymphocyte-associated antigen.</a> J. Clin. Invest. 112, 1008-1018 .</li><li>Dimitroff, C. J., Lechpammer, M., Long-Woodward, D., Kutok, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rolling+of+human+bone-metastatic+prostate+tumor+cells+on+human+bone+marrow+endothelium+under+shear+flow+is+mediated+by+E-selectin.">Rolling of human bone-metastatic prostate tumor cells on human bone marrow endothelium under shear flow is mediated by E-selectin.</a> Cancer Res. 64, 5261-5269 (2004).</li><li>Dimitroff, C. J., Descheny, L., Trujillo, N., Kim, R., Nguyen, V., Huang, W., Pienta, K. J., Kutok, J. L., Rubin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+leukocyte+E-selectin+ligands,+P-selectin+glycoprotein+ligand-1+and+E-selectin+ligand-1,+on+human+metastatic+prostate+tumor+cells.">Identification of leukocyte E-selectin ligands, P-selectin glycoprotein ligand-1 and E-selectin ligand-1, on human metastatic prostate tumor cells.</a> Cancer Res. 65, 5750-5760 (2005).</li><li>Descheny, L., Gainers, M. E., Walcheck, B., Dimitroff, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ameliorating+Skin-Homing+Receptors+on+Malignant+T+cells+with+a+Fluorosugar+Analog+of+N-acetylglucosamine:+P-selectin+Ligand+is+a+More+Sensitive+Target+than+E-selectin+Ligand.">Ameliorating Skin-Homing Receptors on Malignant T cells with a Fluorosugar Analog of N-acetylglucosamine: P-selectin Ligand is a More Sensitive Target than E-selectin Ligand.</a> J Invest Dermatol. 126 (9), 2065-2073 (2006).</li><li>Gainers, M. E., Descheny, L., Barthel, S. B., Liu, L., Wurbel, M., Dimitroff, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin-homing+receptors+on+effector+leukocytes+are+differentially+sensitive+to+glyco-metabolic+antagonism+in+allergic+contact+dermatitis.">Skin-homing receptors on effector leukocytes are differentially sensitive to glyco-metabolic antagonism in allergic contact dermatitis.</a> J. Immunology. 179 (12), 8509-8518 (2007).</li><li>Rood, P. M., Calafat, J., von dem Borne, A. E., Gerritsen, W. R., van der Schoot, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+human+bone+marrow+endothelial+cells:+characterisation+of+new+cell+lines.">Immortalization of human bone marrow endothelial cells: characterisation of new cell lines.</a> Eur J Clin Invest. 30 (7), 618-629 (2000).</li></ol>

Los autores no tienen conflictos de intereses o revelaciones.

De placas paralelas análisis de la cámara de flujo es un organismo especializado en el sistema de ensayo in vitro utilizados para el estudio de leucocitos - interacciones endotelio adhesivo bajo condiciones de estrés de corte similares, constituidos en la superficie de una vénula post-capilar. Como se muestra aquí, en nuestra presentación visual, que demuestran el valor de este sistema para investigar el papel de E-selectina - E-selectina ligandos en la mediación de la inmovilización de leucocitos y rodar sobre la superficie del endotelio microvascular activado. También revelan la aplicación de la proteasa, glicosidasa y el bloqueo de tratamientos con anticuerpos para dilucidar el papel de la E-selectina y sus ligandos en este sistema. Estos análisis son altamente reproducibles y ayudan a formar una base experimental para el estudio de E-selectina - E-selectina ligando la función in vivo. Además de estudiar la adhesión celular con monocapas de citocinas activa las células endoteliales microvasculares (vena umbilical hBMEC o humanos o células endoteliales microvasculares dérmicas), los ensayos se pueden realizar utilizando humano recombinante purificado E-selectina y E-selectina-Ig moléculas quiméricas como sustrato para la adhesión celular. Otras interacciones adhesivas mediada a través de los leucocitos (L)-selectina y de plaquetas (P)-selectina también se puede probar usando la tecnología de placas paralelas cámara de flujo. Al variar el tipo de monocapa de células / sustrato de la proteína o de leucocitos de entrada /-selectina transfectante modelos de células, los investigadores pueden estudiar el papel de estas interacciones fisiológicas bajo condiciones de esfuerzo cortante.

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>F6178-500ML</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14190</td>
<td>&nbsp;</td>
<tr>
<td>0.5 M EDTA</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15575-038</td>
<td>&nbsp;</td>
<tr>
<td>1M HEPES</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Parallel-Plate Flow Chamber Kit</td>
<td>&nbsp;</td>
<td>GlycoTech</td>
<td>31-001</td>
<td>&nbsp;</td>
<tr>
<td>PHD2000 Programmable Syringe Pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>PHD 2000</td>
<td>&nbsp;</td>
<tr>
<td>35x10mm Tissue Culture Dish</td>
<td>&nbsp;</td>
<td>BD FALCON</td>
<td>353001</td>
<td>&nbsp;</td>
<tr>
<td>Fibronectin , from human plasma</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>86088-83-7 F2006-2MG</td>
<td>&nbsp;</td>
<tr>
<td>Interleukin-1_, Human; Recombinant</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>I9401-5UG</td>
<td>&nbsp;</td>
<tr>
<td>HBSS (Hank s Buffered Saline Solution)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14170</td>
<td>&nbsp;</td>
<tr>
<td>Medium 199 w/ HEPES & Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12320-039</td>
<td>&nbsp;</td>
<tr>
<td>Human Serum</td>
<td>&nbsp;</td>
<td>Innovative Research, Inc.</td>
<td>IPLA-SER1</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Abraxis Pharmaceutical Products</td>
<td>504011</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant human fibroblast growth factor</td>
<td>&nbsp;</td>
<td>R&D Systems</td>
<td>233-FB</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin Streptomycin</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Plastic Test tubes</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>352008</td>
<td>&nbsp;</td>
<tr>
<td>Anti-human E-selectin clone BBIG-E4(5D11)</td>
<td>&nbsp;</td>
<td>R&D Systems	</td>
<td>BBA16</td>
<td>&nbsp;</td>
<tr>
<td>Bromelain</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>B-4882</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
				 	
	

		</div>

analysis of physiologic e-selectin-mediated leukocyte rolling on microvascular endothelium

E-selectina es una proteína de tipo-1 de membrana en las células endoteliales microvasculares que ayuda a iniciar el reclutamiento de leucocitos circulantes a los tejidos cutáneos, óseos y se inflama. E-selectina expresión es constitutiva en la microvasculatura dérmica y ósea y es inducible por citoquinas pro-inflamatorias, como la IL-1α / y el TNF-α, en la microcirculación en los tejidos inflamados. Este receptor de lectina media débiles interacciones de enlace con los hidratos de carbono de los receptores contra los ligandos en los leucocitos circulantes, lo que resulta en un comportamiento característico de laminación. Debido a estas interacciones preceden a los sucesos adhesivo más estable y la actividad diapedesis, la caracterización de la actividad de los leucocitos de laminación y la identificación de leucocitos E-selectina ligandos han sido objetivos principales en los estudios de tráfico de leucocitos y la inflamación y en el desarrollo de terapias anti-inflamatorios (1-5) . La intención de este informe es facilitar una descripción detallada y completa de la tecnología más utilizada para el estudio de E-selectina E-selectina ligando bajo condiciones fisiológicas del flujo sanguíneo. Nuestro laboratorio en colaboración con el Centro de Investigación de Enfermedades de piel de Harvard utiliza una tecnología de última generación de placas paralelas aparato de la cámara de flujo acompañado de visualización digital y software de grabación de nuevo, NIS-Elements. Esta tecnología nos permite analizar eventos de adhesión en tiempo real para la visualización en pantalla, así como registrar la actividad de laminación en un formato de vídeo. Parámetros de adhesión celular, como de frecuencia de paso, la resistencia al corte y unión / inmovilización de eficiencia, se calculan con NIS-Elements, exportados a una hoja de cálculo Excel y sometidos a análisis estadístico. En la demostración de que aquí se presenta, se empleó la cámara de flujo de placas paralelas para investigar la E-selectina dependiente de la actividad de leucocitos rodar en células humanas vivas de la médula ósea del endotelio (hBMEC). Células hematopoyéticas humanas KG1a progenitoras, que expresan un alto nivel de E-selectina ligando, se utilizaron como modelo de leucocitos, mientras que una línea de células inmortalizadas hBMEC, HBMEC-60 células, se utilizó como modelo de las células endoteliales (6). Para inducir y simular nativos E-selectina expresión en la cámara de flujo, HBMEC-60 células se activan en primer lugar con IL-1. Nuestra presentación en video mostró que de placas paralelas análisis de flujo es un método adecuado para el estudio fisiológico de E-selectina leucocitaria mediada por las actividades de laminación y que la caracterización funcional de los leucocitos E-selectina ligando (s) en la cámara de flujo puede determinarse mediante la aplicación de la proteasa o glicosidasas digestiones.

Watch this Scientific Journal Video about Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium at JoVE.com

Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

Este informe ofrece una descripción visual de análisis de placas paralelas cámara de flujo para el estudio de las interacciones leucocito endotelial bajo una tensión cortante fisiológico. Este método es especialmente útil para investigar la función del endotelio (E)-selectina y E-selectina leucocitaria ligandos que activar leucocitos rodar sobre la superficie de las células endoteliales.

Análisis de los Rolling fisiológicos de E-selectina leucocitaria mediada por el endotelio microvascular

E-selectin is a type-1 membrane protein on microvascular endothelial cells that helps initiate recruitment of circulating leukocytes to cutaneous, bone ...

analysis-physiologic-e-selectin-mediated-leukocyte-rolling-on

Research

JoVE Journal

Biology

19.0K vistas.  Brigham and Women's Hospital. Este informe ofrece una descripción visual de análisis de placas paralelas cámara de flujo para el estudio de las interacciones leucocito endotelial bajo una tensión cortante fisiológico. Este método es especialmente útil para investigar la función del endotelio (E)-selectina y E-selectina leucocitaria ligandos que activar leucocitos rodar sobre la superficie de las células endoteliales.

Video: Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A neuronales y astrocitos Co-cultivo de ensayo para el análisis de alto contenido de neurotoxicidad

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Investigación

Biología

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

Bioingeniería Humanos redes microvasculares en ratones inmunodeficientes

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

RNA-seq Análisis de transcriptomes en trombina tratamiento y de control pulmonar humana células endoteliales microvasculares

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

Aislamiento de microvascular endotelial Tubos de arterias de resistencia Ratón

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Medición y Análisis de la producción de ácido extracelular para Determinar glicolítica Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Cuantificación de proteína específica lisina acetilación y Succinylation estequiometría usando Spectrometry total de la adquisición de datos-independiente

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Expresión de proteínas fluorescentes de fusión en murinas médula ósea células dendríticas y macrófagos

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Medidas simultáneas de calcio intracelular y el potencial de membrana en el endotelio Cerebral de ratón recién aislado e intacto

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In vitro Análisis de la función E3 Ubiquitina Ligasa

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.