Este trabajo fue apoyado por el Departamento de Anestesia y Cuidados perioperatorios UCSF.

1. Revestimiento y mantenimiento de las células endoteliales microvasculares <ol><li> Descongele y hacer crecer las células endoteliales microvasculares de acuerdo a los fabricantes suministran instrucciones. En este protocolo, las células fueron cultivadas en medio EGM-2 MV (Lonza), y se recomienda que todos los experimentos se llevan a cabo dentro de dos semanas de descongelación para minimizar cualquier variación debida al número de pases. Nuestros experimentos con HMVEC-pulmón se llevan a cabo entre los pasos 4 a 9. </li><li> Día 1: Cuando las células alcanzan 80-90% de confluencia, trypsinize y resuspender en 120.000 células por ml. Placa de 36.000 células (0,3 ml) por pocillo en 48 pocillos, placa de cultivo tisular de poliestireno (s). Incluir pozos de HMVEC que no se incubaron con neutrófilos con el fin de obtener una lectura de fluorescencia de fondo. A menos que el uso de placas de 48 pocillos diseñadas específicamente para ensayos de fluorescencia, filas o columnas alternas será minimizar cualquier contaminación lumínica de los pozos vecinos. NOTA: Los pozos pueden ser pre-recubiertas con poli-Lysine, colágeno o fibronectina. </li><li> Día 2: Agregar medio fresco. </li><li> Día 3: Por microscopía de contraste de fase, confirman las células están sanos (es decir, la apariencia &quot;adoquines&quot; y poco restos de células), y son 100% confluentes (Figura 1). Si las células no son 100% confluentes, cambiar los medios de comunicación todos los días hasta que estén listos. </li><li> Una vez que las monocapas HMVEC están listos para el tratamiento, lavar las células una vez con solución salina equilibrada de Hank (HBSS) y añadir 0,3 ml de EGM-2 MV medios frescos o medios que contienen agonista inflamatoria a los pocillos apropiados. En este protocolo la HMVEC-pulmón fueron tratados con TNF [100 ng / ml] (PeproTech, Nueva Jersey) durante 3 horas ya que este es un activador bien descrito de ECs 21. NOTA: Se recomienda que el tiempo de su experimento para que sus células HMVEC activados (Paso 4.1) y calceína AM neutrófilos marcados (Paso 3.5) están listos para su uso al mismo tiempo. Nos lleva aproximadamente 2,5 horas para aislar y etiquetar neutrophils. </li></ol> 2. Aislamiento de neutrófilos mediante Polymorphprep <ol><li> Aislar los neutrófilos como se ha descrito previamente 22, o con una solución de gradiente de densidad confeccionada para aislar los granulocitos polimorfonucleares de la sangre entera. El empleo de Polymorphprep siguiendo el protocolo por Nuzzi et.al. 2007 resultados de los rendimientos de aproximadamente 2-3 x 10 6 neutrófilos por ml de sangre entera 23. NOTA: Para evitar la lisis de los eritrocitos excesiva, una sedimentación de dextrano para eliminar las células rojas de la sangre puede llevar a cabo antes de la centrifugación en gradiente de densidad 24. </li><li> Poner la solución en gradiente de densidad Polymorphprep a RT. </li><li> Recoger 30 ml de sangre completa de un voluntario humano sano en presencia de un anticoagulante tal como heparina, citrato o EDTA. La sangre se debe utilizar dentro de 2 horas, y se mantiene entre los 18-22 ° C. </li><li> Capa 5 ml de sangre entera sobre 5 ml de la solución de gradiente de densidad en un tubo cónico de 15 ml (Figura 2A). Evite alterar volúmenes de sangre y la solución de gradiente de densidad, ya que podría cambiar las condiciones de centrifugación posteriores. </li><li> Centrifugar a 450 xg durante 30 min a 18-22 ° C. Asegúrese de que para acelerar lentamente arriba y abajo de la centrífuga (es decir, desactivar el freno de centrífuga), y el equilibrio de los tubos así para evitar vibraciones para ayudar a reducir la activación de los neutrófilos y evitar la mezcla de las capas. Tiempos de centrifugación más largos o más alta fuerza centrífuga se traducirá en la capa de leucocitos inferior, que contiene los neutrófilos, a migrar más abajo hacia a las células rojas de la sangre sedimentadas. </li><li> Aspirar el plasma y la banda de leucocitos superior que contiene las PBMC para evitar la contaminación de la capa que contiene los neutrófilos (Figura 2B). </li><li> Con una pipeta serológica 1-2 ml para eliminar la capa inferior que contiene los leucocitos neutrófilos. Se combinan las capas de neutrófilos en 30 ml de PBS (sin Ca 2 + y Mg 2 +) en un 50 mltubo cónico. </li><li> Llevar el volumen hasta 50 ml con PBS (sin Ca 2 + y Mg 2 +) y se centrifuga a 450 g durante 10 min a 18-22 ° C. </li></ol> Nota: Los pasos 2.9 y 2.10 son opcionales, pero altamente recomendable. <ol start="9"><li> Aspirar el sobrenadante. Resuspender los neutrófilos en 8 ml de agua estéril durante 30 segundos para lisar las células rojas de la sangre, añadir inmediatamente la suspensión de células a 2 ml de 5x PBS (sin Ca 2 + y Mg 2 +) y mezclar suavemente con la mano. No incubar las células en agua durante más de 30 segundos desde la muerte de neutrófilos significativa puede ocurrir. </li><li> Centrifugar a 250 xg durante 5 min a 18-22 ° C. </li><li> Se lavan las células una vez con 10 ml de PBS (sin Ca 2 + y Mg 2 +) y centrifuga de nuevo a 250 xg durante 5 min a 18-22 ° C. </li><li> Resuspender los neutrófilos en 10 ml de medio RPMI-1640 (sin rojo fenol) y contar las células vivas utilizando el azul de tripanoteñir método de exclusión. Evitar períodos prolongados de densidades celulares superiores a 5 x 10 6 por ml ya que esto puede resultar en la activación de los neutrófilos. </li></ol> 3. Neutrófilos Etiquetado con calceína AM <ol><li> 6 x 10 5 neutrófilos se utilizan por pocillo de una placa de 48 pocillos. </li><li> Después de contar, transferir las células resuspendidas a un tubo cónico de 50 ml y diluir los neutrófilos a 2-4 x 10 6 células por ml con rojo fenol libre de RPMI-1640. </li><li> Añadir calceína AM solución madre de 3 m (es decir 2,98 l de calceína AM stock (1 mg / ml) por ml de medio) y mezclar bien accionando suavemente el tubo. Mayor que 5 mM calceína AM dará lugar a fugas de los neutrófilos Nota:. No fluorescente calceína AM se escinde dentro de las células por las esterasas endógenas para producir la molécula altamente fluorescente calceína (es decir, las células muertas no fluorescencia). </li><li> Cubra el tubo con papel de aluminio e incubar for 30 min a 37 ° C en la oscuridad Nota:. tiempos de incubación más largos puede resultar en más calceína ser liberado de los neutrófilos en el transcurso del ensayo de adhesión. </li><li> Centrifugar a 450 xg durante 5 min a 18-22 ° C, y lavar los neutrófilos 2x con 10 ml de medio RPMI-1640 (sin rojo de fenol; pre-calentado a 37 ° C). Después de resuspender las células para el segundo lavado, primero pasar a los neutrófilos a través de un filtro estéril 40 mM para eliminar grumos y, a continuación centrifugar una vez más a 450 xg durante 5 min a 18-22 ° C. </li><li> Resuspender los neutrófilos en medio RPMI 1640 (sin rojo de fenol; pre-calentado a 37 ° C) a 2 x 10 6 neutrófilos por ml. </li></ol> 4. Ensayo de unión de neutrófilos <ol><li> Se lavan las monocapas HMVEC 3x con 0,5 ml de esterilizada por filtración (0,22 micras) RPMI 1640 (sin rojo fenol) que contiene 3% de BSA por pocillo. </li><li> Aspirar los medios de comunicación y añadir 6 x 10 5 neutrófilos marcados con calceína-(0,3 ml de suspensión de células), O 0,3 ml de medio RPMI 1640 (sin rojo fenol) sin neutrófilos, a los pocillos apropiados de la placa de 48 pocillos Nota:. Este es el equivalente de 8 x 10 5 neutrófilos por cm 2. </li><li> Incubar durante 20 min en un 37 ° C, 5% de CO 2 incubadora. </li><li> Total de neutrófilos fluorescencia (PRE lavado): Mide la intensidad de fluorescencia de cada pocillo con una longitud de onda de excitación de 485 nm y una longitud de onda de emisión de 520 nm (filtro establecido fluoresceína) en una FLUOstar, o equivalente, espectrofotómetro. Realice este paso justo antes del punto final de incubación Nota: En este protocolo, calceína de excitación y detección de la luz emitida se realizaron a partir de la parte superior de los pozos no cubierto, sin embargo, ambos también se puede realizar desde el fondo de los pocillos.. </li><li> Lave los pocillos que contienen monocapas HMVEC y neutrófilos 5x con 0,5 ml por pocillo de PBS (w / Ca 2 + y Mg 2 +). Retire el papel y lavadosinvirtiendo la placa, y palmaditas suavemente sobre toallas de papel para eliminar el líquido restante. </li><li> Añadir nuevo 0,3 ml de RPMI 1640 (sin rojo fenol) por pocillo. </li><li> Adherente de neutrófilos fluorescencia (POST lavado): Medir la intensidad de fluorescencia de cada pocillo como en el paso 4.4. </li><li> Determinar el porcentaje de cumplimiento y la adherencia relativa por así: </li></ol> Nota: En esta etapa, las imágenes de los neutrófilos marcados con calceína se puede conseguir mediante el uso de un microscopio capaz de fluorescencia equipado con filtros de fluoresceína estándar. Las imágenes en la Figura 4 se obtuvieron en una Olympus IX51 microscopio invertido equipado con una cámara 2000R Retiga (Q Imaging) utilizando Q-Capture Pro7 software (Q Imaging).

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U.S.A. 82, 8667-8671 (1985).</li><li>Braut-Boucher, F., 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=A+non-isotopic,+highly+sensitive,+fluorimetric,+cell-cell+adhesion+microplate+assay+using+calcein+AM-labeled+lymphocytes.">A non-isotopic, highly sensitive, fluorimetric, cell-cell adhesion microplate assay using calcein AM-labeled lymphocytes.</a> Journal of Immunological Methods. 178, 41-51 (1995).</li><li>Ait-Oufella, H., Maury, E., Lehoux, S., Guidet, B., Offenstadt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelium:+physiological+functions+and+role+in+microcirculatory+failure+during+severe+sepsis.">The endothelium: physiological functions and role in microcirculatory failure during severe sepsis.</a> Intensive Care Medicine. 36, 1286-1298 (2010).</li><li>Andonegui, 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=Endothelium-derived+Toll-like+receptor-4+is+the+key+molecule+in+LPS-induced+neutrophil+sequestration+into+lungs.">Endothelium-derived Toll-like receptor-4 is the key molecule in LPS-induced neutrophil sequestration into lungs.</a> The Journal of Clinical Investigation. 111, 1011-1020 (2003).</li><li>Sharma, J., 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=Lung+endothelial+cell+platelet-activating+factor+production+and+inflammatory+cell+adherence+are+increased+in+response+to+cigarette+smoke+component+exposure.">Lung endothelial cell platelet-activating factor production and inflammatory cell adherence are increased in response to cigarette smoke component exposure.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 302, L47-L55 (2012).</li><li>Deban, L., Correale, C., Vetrano, S., Malesci, A., Danese, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pathogenic+roles+of+microvasculature+in+inflammatory+bowel+disease:+a+Jack+of+all+trades.">Multiple pathogenic roles of microvasculature in inflammatory bowel disease: a Jack of all trades.</a> The American Journal of Pathology. 172, 1457-1466 (2008).</li><li>Gross, W. L., Trabandt, A., Csernok, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Wegener&amp;#39;s+granulomatosis.">Pathogenesis of Wegener&amp;#39;s granulomatosis.</a> Annales de Medecine Interne. 149, 280-286 (1998).</li><li>Chen, Y., 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=Evidence+of+inflammatory+cell+involvement+in+brain+arteriovenous+malformations.">Evidence of inflammatory cell involvement in brain arteriovenous malformations.</a> Neurosurgery. 62, 1340-1349 (2008).</li><li>Martens, C. 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=Peptides+which+bind+to+E-selectin+and+block+neutrophil+adhesion.">Peptides which bind to E-selectin and block neutrophil adhesion.</a> J. Biol. Chem. 270, 21129-21136 (1995).</li></ol>

Los autores tienen nada que revelar.

Los pasos más importantes para el éxito de neutrófilos / ensayo de adhesión celular endotelial microvascular son los siguientes: 1) El uso del número de paso bajo (&lt;9), células endoteliales sanas, 2) El mantenimiento de los neutrófilos aislados con una densidad baja (es decir, células &lt;5 x 10 6 / ml) y el uso de ellos en las dos horas de aislamiento, y 3) el lavado Fastidious de los neutrófilos calceína AM-etiquetados y el uso de los neutrófilos calceína AM-etiquetados de manera opo...

Dado que el endotelio vascular está en contacto directo con la sangre circulante, que tiene una ubicación única para iniciar una respuesta inflamatoria rápida durante la infección o lesión. Las células endoteliales (EC) receptores de reconocimiento de patrones expresas que reconocen una variedad de componentes bacterianos conservados y las moléculas de peligro, y los receptores para sistemas principales mediadores de la inflamación tales como TNFa. La activación de estos receptores induce ECs para secretar citoquinas (por ejemplo, IL-6, IL-8, CXCL1 y CCL2), y para upregulate moléculas de adhesión (por ejemplo, E / P-selectina, VCAM-1 e ICAM-1) en su superficie celular 1 , 2. Estas moléculas de todo facilitar la localización de los leucocitos a los sitios de infección y la lesión con el fin de despejar el campo de los agentes infecciosos e iniciar la reparación del tejido 3,4. La respuesta de los neutrófilos a la infección implica una interacción bien coordinada entre el endotelio vascular y la respuesta los neutrófilos. Tras la activación CE, IL-8 se secreta y forma un gradiente intravascular en el endotelio que permite a los neutrófilos a la casa en el sitio de la infección o lesión 5,6. E / P-selectinas median la captura de neutrófilos y rodando a través de asociaciones relativamente débiles con glycomolecules en la superficie celular de neutrófilos. Estas interacciones, junto con la IL-8 la unión a sus receptores afines, facilitan la robusta, unión mediada por la integrina y la eventual detención de los neutrófilos sobre la superficie de la célula endotelial 7-10. Después de la detención, los neutrófilos pueden migrar fuera de la vasculatura a los sitios específicos de infección para eliminar directamente los patógenos, generar trampas extracelulares de neutrófilos para prevenir la propagación de los agentes patógenos, promover la cicatrización de heridas y liberar factores adicionales que reclutan otros leucocitos tales como monocitos, macrófagos y dendríticas 11-17 células. Se describe en el presente informe es un método in vitro para cuantificar la adhesión de neutrófilos a microVascular ECs después de la activación por el mediador inflamatorio TNF anfitrión. Este ensayo está diseñado para evaluar la activación de EC, y no los neutrófilos. Los neutrófilos humanos primarios son primero aislados utilizando separación por gradiente de densidad, y luego se marcan con calceína acetoximetilo (AM). Esterasas dentro de las células en vivo hidrolizan calceína AM a la molécula calceına altamente fluorescente con una excitación de 492-495 nm y de emisión de 513-516 nm 18. Los neutrófilos marcados con fluorescencia se incuban a continuación con monocapas CE, y los neutrófilos no adherentes se eliminan posteriormente. La fluorescencia de las restantes, neutrófilos enlazados a continuación, se mide utilizando un espectrofotómetro de fluorescencia, y se calcula como un porcentaje del total de entrada de la fluorescencia de neutrófilos por pocillo. Este método tiene la ventaja adicional de que los neutrófilos calceína-marcado unido utilizados en espectrofotometría se pueden visualizar directamente mediante microscopía de fluorescencia para dar una más cualitativa lectura de CE de corriente alternavación. Puesto que este ensayo se lleva a cabo bajo condiciones estáticas, únicamente los eventos muy iniciales que se producen en la cascada de adhesión de neutrófilos serán evaluados. Esto se confirma en el informe utilizando anticuerpos bloqueantes E-selectina para mostrar que la adhesión de neutrófilos al pulmón humano TNF-tratada microvascular CE (HMVEC-pulmón) monocapas se reduce drásticamente cuando se interrumpe la interacción con E-selectina. Además de TNFa, hemos utilizado con éxito este ensayo para determinar la extensión de la vena activación CE umbilical humana (HUVEC) por el receptor de tipo Toll medio agonistas lipoproteína asociada a peptidoglicano (PAL), mureína lipoproteína (MLP) y Pam3Cys y activación HMVEC-Pulmón por Pam3Cys 19,20. Además, hemos utilizado con éxito este ensayo con inhibidores de la quinasa y después de RNAi mediada desmontables de superficie y proteínas citoplasmáticas en HMVEC-pulmón, lo que sugiere que esta metodología es compatible con una variedad de bioquímica y screeninensayos de 20 g. En resumen, este ensayo proporciona una fácil de usar, forma reproducible, más funcional para acceder a la medida de la activación de CE por mediadores inflamatorios in vitro.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>HMVEC-Lung</td>
 <td>Lonza</td>
 <td>CC-2527</td>
 <td></td>
 </tr>
 <tr>
 <td>EGM-2 MV</td>
 <td>Lonza</td>
 <td>CC-3202</td>
 <td></td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Life Technologies</td>
 <td>14175-095</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>48-well Tissue Culture Plates</td>
 <td>BD Falcon</td>
 <td>353078</td>
 <td></td>
 </tr>
 <tr>
 <td>PBS (without phenol red)</td>
 <td>UCSF Cell Culture Facility</td>
 <td>CCFAL001 </td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>D-PBS (without Ca2+ and Mg2+ and phenol red)</td>
 <td>UCSF Cell Culture Facility</td>
 <td>CCFAL003</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>RPMI-1640 (without phenol red)</td>
 <td>Life Technologies</td>
 <td>11835-030</td>
 <td></td>
 </tr>
 <tr>
 <td>Polymorphprep</td>
 <td>Axis-Shield</td>
 <td>1114683</td>
 <td></td>
 </tr>
 <tr>
 <td>calcein AM</td>
 <td>Life Technologies</td>
 <td>C3099</td>
 <td>(0.995 mM) stock soln</td>
 </tr>
 <tr>
 <td>Trypan Blue Solution</td>
 <td>Sigma-Aldrich</td>
 <td>T8154</td>
 <td></td>
 </tr>
 <tr>
 <td>40 &mu;M filters</td>
 <td>VWR</td>
 <td>21008-949</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>FLUOstar OPTIMA Spectrophotometer</td>
 <td>BMG LABTECH</td>
 <td>-</td>
 <td></td>
 </tr>
 </tbody>
</table>

quantitative in vitro assay to measure neutrophil adhesion to activated primary human microvascular endothelial cells under static conditions

El endotelio vascular juega un papel integral en la respuesta inflamatoria. Durante la fase aguda de la inflamación, las células endoteliales (EC) se activan por los mediadores de acogida o directamente por componentes microbianos conservados peligro o moléculas derivadas del huésped. ECs activados expresan citocinas, quimiocinas y moléculas de adhesión que movilizan, activan y retienen los leucocitos en el sitio de la infección o lesión. Los neutrófilos son las primeras en llegar leucocitos, y se adhieren al endotelio a través de una variedad de moléculas de adhesión presentes en las superficies de ambas células. Las principales funciones de los neutrófilos son para eliminar directamente las amenazas microbianas, promover el reclutamiento de otros leucocitos a través de la liberación de factores adicionales, e iniciar la reparación de la herida. Por lo tanto, su reclutamiento y el apego al endotelio es un paso crítico en la iniciación de la respuesta inflamatoria. En este reporte se describe un ensayo in vitro de la adhesión de neutrófilos mediantecalceína AM marcada con neutrófilos humanos primarios para cuantificar el grado de activación de las células endoteliales microvasculares en condiciones estáticas. Este método tiene la ventaja adicional de que las mismas muestras se cuantificaron por espectrofotometría de fluorescencia también se pueden visualizar directamente mediante microscopía de fluorescencia para una evaluación más cualitativa de la unión de neutrófilos.

Con el fin de obtener resultados fiables, reproducibles utilizando un ensayo de unión de neutrófilos, es esencial que la salud y la confluencia de las células endoteliales microvasculares son óptimas en el día del ensayo, como se ilustra con HMVEC-pulmón en la Figura 1. Además, es imperativo que el número de pase bajo microvascular AE se utiliza (es decir, menos de 9 pasajes), y, en consecuencia, se recomienda llevar a cabo todos los experimentos en las dos semanas siguientes a la desco...

Watch this Scientific Journal Video about Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions at JoVE.com

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

Adherencia de los neutrófilos al endotelio activado en los sitios de infección es un componente integral de la respuesta inflamatoria del huésped. Se describe en el presente informe es un ensayo de unión de neutrófilos, que permite la<em&gt; In vitro</em&gt; Cuantificación de neutrófilos humana primaria unión a las células endoteliales activadas por los mediadores inflamatorios en condiciones estáticas.

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 ...

quantitative-vitro-assay-to-measure-neutrophil-adhesion-to-activated

Research

JoVE Journal

Immunology and Infection

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

17.0K vistas.  University of California, San Francisco. Adherencia de los neutrófilos al endotelio activado en los sitios de infección es un componente integral de la respuesta inflamatoria del huésped. Se describe en el presente informe es un ensayo de unión de neutrófilos, que permite la In vitro Cuantificación de neutrófilos humana primaria unión a las células endoteliales activadas por los mediadores inflamatorios en condiciones estáticas.

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

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 ...

Investigación

Inmunología e Infección

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Aislamiento de células endoteliales de vena umbilical y su uso en el Estudio de la transmigración de neutrófilos bajo condiciones de flujo

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Un<em&gt; In vitro</em&gt; Co-infección modelo para estudiar<em&gt; Plasmodium falciparum</em&gt;-VIH-1 en las interacciones humano primario de monocitos derivados de las células inmunitarias

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

In vitro Ensayo de cocultivo para evaluar la migración transepitelial de neutrófilos inducida por patógenos

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

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 ...

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

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

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 ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

Imágenes en tiempo real de la célula-célula cruces endoteliales Durante neutrófilos Transmigración Bajo Flujo Fisiológica

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

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 ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Ensayo de adhesión en condiciones de estrés de cizalla para el Estudio de linfocitos T-molécula de adhesión Interacciones

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...