Nos gustaría dar las gracias a M. Höxter para la asistencia técnica en la clasificación AECII murino primaria de bioseguridad de nivel 2 muestras. Este trabajo fue apoyado por becas de la Fundación Alemana de Investigación (DFG) a DB (SFB587, B12 y BR2221/1-1 TP) y una beca de la Escuela de Investigación Biomédica de Hannover (DFG GSC 108) a AA. DB con el apoyo de la Iniciativa del Presidente y del Fondo de Redes de la Asociación Helmholtz de Centros de Investigación alemán (HGF), bajo contrato W2/W3-029 número.

Los detalles relativos a los reactivos y materiales necesarios se enumeran en la tabla al final del protocolo a continuación. Antes de empezar el trabajo, preparar tubos de 15 ml (una por ratón) que contienen 4 ml de dispasa y pre-calentamiento a 37 ° C en un baño de agua. En un bloque de calentamiento, poco calentar pequeñas alícuotas de bajo punto de fusión al 1% de agarosa (en agua) a 95 ° C hasta que la miel y, posteriormente, enfriar a 45 ° C hasta su uso. 1. Preparación del pulmón de ratón <ol><li> Sacrificar el ratón por asfixia de CO 2. Nota: No realice dislocación cervical, ya que esto lesionar la tráquea de modo que siguiendo los pasos del protocolo, como la instalación de los pulmones con líquido, no se puede realizar con éxito. Asegúrese de que la pérdida de los reflejos nociceptivos. </li><li> Pulverizar el ratón con etanol y hacer un corte largo a lo largo de la línea media ventral del cuerpo. Tire de la piel y la piel ventral y posteriormente también cuidadosamente cortar y quitar el peritoneo. </li><li> Exsanguinate laratón cortando la vena yugular izquierda y la derecha (Vena yugular), así como la arteria renal (renalis Arteria). Quite la sangre que fluye con el tejido. </li><li> Con cuidado, punción y retire el diafragma para exponer el corazón y los pulmones cortando las costillas. Tenga especial cuidado de no lesionar el pulmón. </li><li> Con una cánula 26G y una jeringa de 10 ml llena con solución salina tamponada con fosfato fría (PBS), la punción del ventrículo derecho del corazón y perfundir el pulmón con PBS hasta que esté libre de sangre. </li><li> Corte y retire las glándulas salivales para exponer la tráquea. También con cuidado corte el músculo que rodea la tráquea. </li><li> Insertar una cánula permanente 22G en la tráquea, retirar la aguja y empujar el catéter de plástico hacia el pulmón. Fijar el catéter mediante la vinculación de una pequeña pieza de lana alrededor de la tráquea y el catéter. </li></ol> 2. La digestión enzimática del tejido pulmonar Si se desea, una muestra de líquido de lavado broncoalveolar puede serpreparado por el lavado de los pulmones a través del catéter insertado en la tráquea con PBS o medio antes de la siguiente etapa. <ol><li> Usando una jeringa de 2 ml, cuidadosamente infundir un volumen máximo de 2 ml de dispasa (de la alícuota ml 4) en el pulmón a través del catéter de modo que todos los lóbulos están totalmente expandido. Intercambiar la jeringa con una jeringa de 1 ml que contiene 0,5 ml de la agarosa licuada y también infundir en el pulmón. </li><li> Deje la jeringa en el catéter para evitar el flujo de retorno de la agarosa y cubrir inmediatamente el pulmón con papel de laboratorio del tejido y hielo. Deje que el gel de agarosa durante un par de minutos. </li><li> Quitar el papel de seda, hielo, jeringas y catéteres, cortar la tráquea y escindir el pulmón, corazón y timo del pecho. Luego enjuague el pulmón en un plato con PBS y quitar el corazón, el timo y la tráquea restante. </li><li> Ponga el pulmón hacia el restante 2 ml de dispasa y se incuba a temperatura ambiente durante 45 min. </li></ol> 3. Preparación del pulmónSuspensión celular <ol><li> Retire el pulmón de la dispasa y la transfiere en un plato que contiene 7 ml de anticuerpo anti-CD16/32 (1 mg / ml concentración final) en medio DMEM y 100 l DNasa. </li><li> Utilizando fórceps, se desintegran completamente el tejido pulmonar tirando de él aparte y se incuba durante 10 min a temperatura ambiente mientras agitando suavemente en un agitador ajustado a 200 rpm. Si se desea, la suspensión de células se pueden almacenar a 4 º C hasta el siguiente paso. </li><li> Se filtra la suspensión celular a través de mallas de nylon primero con 100 micras y 70 micras, posteriormente con 48 micras y 30 micras poros. Tomar cuidado de enjuagar cada malla, así como los tubos y las placas concienzudamente con DMEM para minimizar la pérdida de células y maximizar el rendimiento. Si está puesta en común de muestras, esto se puede lograr aquí pasando posteriormente por suspensiones de células de ratones de un grupo a través del mismo filtro. Cambiar el filtro en caso de obstrucción. </li><li> Dependiendo del volumen, transferir el filtrado a uno o más tubos de 50 ml ycentrifugar durante 15 min a 160 xg y 4 ° C. Eliminar el sobrenadante. </li><li> Resuspender el pellet de células en 2 ml de tampón de lisis de eritrocitos (0,15 M NH 4 Cl, 0,01 M de KHCO 3, 0,1 mM EDTA, pH 7,2) y terminar rápidamente la lisis por adición de 13 ml de medio DMEM. Transferir la solución a un tubo de 15 ml y centrifugar durante 12 min a 160 xg y 4 ° C. </li></ol> 4. Antibody tinción para citometría de flujo en células de clasificación <ol><li> Resuspender las células en 3 ml de cóctel de anticuerpos primaria, preparado en medio DMEM en diluciones adecuadas para la citometría de flujo. (Los anticuerpos fueron ajustado previamente para diluciones de trabajo óptimas mediante la tinción de 1 x 10 6 esplenocitos en un volumen de 100 l. Uso 3 ml del cóctel de anticuerpo primario que contiene las concentraciones óptimas para cada uno de los anticuerpos utilizados para células agrupadas de hasta cinco ratones. Si más ratones se agruparon para una muestra, ajustar el volumen). </li><li> Para la tinción, incubar las células durante 10 min en la oscuridada 4 ° C. </li><li> Se lavan las células por el tubo de llenado 15 ml con medio DMEM y centrifugación durante 12 min a 160 xg y 4 º C. </li><li> En el caso de anticuerpos biotinilados o no acoplados se utilizaron en 4,1: Eliminar el sobrenadante y resuspender las células en 2 a 3 ml de cóctel de anticuerpo secundario. Teñir durante 10 min a 4 ° C en la oscuridad. </li><li> Se lavan las células a través de rellenar el tubo con DMEM y centrifugación durante 12 min a 160 xg y 4 ° C. </li><li> Resuspender las células en 1 ml de DMEM y pre-filtro a través de un filtro de 50 micras en un tubo para la clasificación de células. Opcional: Contar las células para obtener el número total de células en la suspensión de células de pulmón. </li></ol> 5. De clasificación de células <ol><li> Mezclar las células mediante agitación en vórtex antes de la clasificación. Use una boquilla de 100 micras para la célula de clasificación de AECII. La presión de fluido de funda, así como de emisión láser y longitudes de onda de detección dependen del instrumento utilizado para la citometría de flujo de clasificación de células, así como los fluorocromos utilizados en la antibody procedimiento de tinción. </li><li> Puerta en las células SSC altos que son negativos para los fluorocromos utilizados para la tinción y ordenar en un tubo que contiene DMEM. Utilice altura dispersión hacia adelante (FSC-H) vs área (FSC-A) y la altura de dispersión lateral (SSC-H) vs área (SSC-A) de Windows para excluir los dobletes o agregados celulares (ver detalles de la estrategia de puerta en Figura 1 a). </li><li> Con el fin de recoger la centrífuga ordenados células durante 20 min a 280 xg y 4 º C. </li><li> Resuspender las células en medio de cultivo o tampón como se requiere para su posterior análisis. </li></ol>

<ol>
	<li>Fehrenbach, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+epithelial+type+II+cell:+defender+of+the+alveolus+revisited.">Alveolar epithelial type II cell: defender of the alveolus revisited.</a> Respir. Res. 2, 33-46 (2001).</li><li>Folkerts, G., Nijkamp, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+epithelium:+more+than+just+a+barrier.">Airway epithelium: more than just a barrier.</a> Trends Pharmacol. Sci. 19, 334-341 (1998).</li><li>Gereke, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+alterations+in+type+II+alveolar+epithelial+cells+in+CD4++T+cell+mediated+lung+inflammation.">Phenotypic alterations in type II alveolar epithelial cells in CD4+ T cell mediated lung inflammation.</a> Respir. Res. 8, 47 (2007).</li><li>Gereke, M., Jung, S., Buer, J., Bruder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+type+II+epithelial+cells+present+antigen+to+CD4(+)+T+cells+and+induce+Foxp3(+)+regulatory+T+cells.">Alveolar type II epithelial cells present antigen to CD4(+) T cells and induce Foxp3(+) regulatory T cells.</a> Am. J. Respir. Crit Care Med. 179, 344-355 (2009).</li><li>Gribar, S. C., Richardson, W. M., Sodhi, C. P., Hackam, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+longer+an+innocent+bystander:+epithelial+toll-like+receptor+signaling+in+the+development+of+mucosal+inflammation.">No longer an innocent bystander: epithelial toll-like receptor signaling in the development of mucosal inflammation.</a> Mol. Med. 14, 645-659 (2008).</li><li>Herold, S., 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=Alveolar+epithelial+cells+direct+monocyte+transepithelial+migration+upon+influenza+virus+infection:+impact+of+chemokines+and+adhesion+molecules.">Alveolar epithelial cells direct monocyte transepithelial migration upon influenza virus infection: impact of chemokines and adhesion molecules.</a> J. Immunol. 177, 1817-1824 (2006).</li><li>Knight, D. A., Holgate, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+airway+epithelium:+structural+and+functional+properties+in+health+and+disease.">The airway epithelium: structural and functional properties in health and disease.</a> Respirology. 8, 432-446 (2003).</li><li>Schmiedl, A., Kerber-Momot, T., Munder, A., Pabst, R., Tschernig, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+distribution+in+lung+parenchyma+early+after+pulmonary+infection+with+Pseudomonas+aeruginosa.">Bacterial distribution in lung parenchyma early after pulmonary infection with Pseudomonas aeruginosa.</a> Cell Tissue Res. 342, 67-73 (2010).</li><li>Loveday, E. K., Svinti, V., Diederich, S., Pasick, J., Jean, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal-+and+Strain-Specific+Host+MicroRNA+Molecular+Signatures+Associated+with+Swine-Origin+H1N1+and+Avian-Origin+H7N7+Influenza+A+Virus+Infection.">Temporal- and Strain-Specific Host MicroRNA Molecular Signatures Associated with Swine-Origin H1N1 and Avian-Origin H7N7 Influenza A Virus Infection.</a> J. Virol. 86, 6109-6122 (2012).</li><li>Marriott, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1beta+regulates+CXCL8+release+and+influences+disease+outcome+in+response+to+Streptococcus+pneumoniae,+defining+intercellular+cooperation+between+pulmonary+epithelial+cells+and+macrophages.">Interleukin-1beta regulates CXCL8 release and influences disease outcome in response to Streptococcus pneumoniae, defining intercellular cooperation between pulmonary epithelial cells and macrophages.</a> Infect. Immun. 80, 1140-1149 (2012).</li><li>Mata, M., Morcillo, E., Gimeno, C., Cortijo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-acetyl-L-cysteine+(NAC)+inhibit+mucin+synthesis+and+pro-inflammatory+mediators+in+alveolar+type+II+epithelial+cells+infected+with+influenza+virus+A+and+B+and+with+respiratory+syncytial+virus+(RSV).">N-acetyl-L-cysteine (NAC) inhibit mucin synthesis and pro-inflammatory mediators in alveolar type II epithelial cells infected with influenza virus A and B and with respiratory syncytial virus (RSV).</a> Biochem. Pharmacol. 82, 548-555 (2011).</li><li>Zarbock, R., 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=The+surfactant+protein+C+mutation+A116D+alters+cellular+processing,+stress+tolerance,+surfactant+lipid+composition,+and+immune+cell+activation.">The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation.</a> BMC. Pulm. Med. 12, 15 (2012).</li><li>Taubenberger, J. K., Morens, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathology+of+influenza+virus+infections.">The pathology of influenza virus infections.</a> Annu. Rev. Pathol. 3, 499-522 (2008).</li><li>Dobbs, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+alveolar+type+II+cells.">Isolation and culture of alveolar type II cells.</a> Am. J. Physiol. 258, L134-L147 (1990).</li><li>Corti, M., Brody, A. R., Harrison, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+primary+culture+of+murine+alveolar+type+II+cells.">Isolation and primary culture of murine alveolar type II cells.</a> Am. J. Respir. Cell Mol. Biol. 14, 309-315 (1996).</li><li>Beers, M. F., Kim, C. Y., Dodia, C., Fisher, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization,+synthesis,+and+processing+of+surfactant+protein+SP-C+in+rat+lung+analyzed+by+epitope-specific+antipeptide+antibodies.">Localization, synthesis, and processing of surfactant protein SP-C in rat lung analyzed by epitope-specific antipeptide antibodies.</a> J. Biol. Chem. 269, 20318-20328 (1994).</li><li>Phelps, D. S., Floros, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+pulmonary+surfactant+proteins+using+immunohistochemistry+and+tissue+in+situ+hybridization.">Localization of pulmonary surfactant proteins using immunohistochemistry and tissue in situ hybridization.</a> Exp. Lung Res. 17, 985-995 (1991).</li><li>Wang, 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=Differentiated+human+alveolar+type+II+cells+secrete+antiviral+IL-29+(IFN-lambda+1)+in+response+to+influenza+A+infection.">Differentiated human alveolar type II cells secrete antiviral IL-29 (IFN-lambda 1) in response to influenza A infection.</a> J. Immunol. 182, 1296-1304 (2009).</li><li>Wang, 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=Innate+immune+response+to+influenza+A+virus+in+differentiated+human+alveolar+type+II+cells.">Innate immune response to influenza A virus in differentiated human alveolar type II cells.</a> Am. J. Respir. Cell Mol. Biol. 45, 582-591 (2011).</li></ol>

No hay conflictos de interés declarado.

El protocolo para el aislamiento de murino AECII por citometría de flujo ofrece una forma rápida de acceder a las células primarias del pulmón de ratón para toda una serie de estudios funcionales y moleculares. El procedimiento descrito se obtiene poblaciones altamente viables y puro de AECII que son suficientes en número para directos análisis posteriores, tales como el aislamiento de ARN (ver figura 2b) y transcriptome estudios. Para aplicaciones funcionales, también es posible cultivar las c?...

<table border="1"><tbody><tr><td> Nombre de reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> mora cánula 22G Introcan </td><td> Braun </td><td> REF 4252098B </td><td></td></tr><tr><td> Dispasa, 100 ml (5000 unidades caseinolíticas) </td><td> BD Biosciences </td><td> 354235 </td><td> alícuota de 4 ml en tubos de 15 ml, almacenar a -20 ° C </td></tr><tr><td> Biozym placa de agarosa </td><td> Biozym </td><td> 840101 </td><td> 1% w / v en H 2 O </td></tr><tr><td> Desoxirribonucleasa I de páncreas bovino, 2000 unidades Kunitz / vial </td><td> Sigma-Aldrich </td><td> D4263 </td><td> recién disolver el contenido de 1 vial en 300 l de DMEM </td></tr><tr><td> DMEM </td><td> Gibco </td><td> 22320-022 </td><td> utilizado según lo previsto por el fabricante (Low Glucose, piruvato, HEPES) </td></tr><tr><td> coladores celulares (100 micras, 75 micras) </td><td> BD Falcon </td><td> 352360, 352350 </td><td></td></tr><tr><td> de malla de nylon (48 micras, 30 micras) </td><td> Buckmann GmbH </td><td> 03-48/26-1020, 03-30/18-108 </td><td></td></tr><tr><td> CellTrics 50 micras filtro </td><td> PARTEC </td><td> 04-0042-2317 </td><td></td></tr><tr><td> anti-ratón CD16/CD32 </td><td> BioLegend </td><td> 101302 </td><td> clon 93; purificada </td></tr><tr><td> anti-ratón de F4/80 </td><td> BioLegend </td><td> 123116 </td><td> clon BM8; APC acoplado </td></tr><tr><td> anti-ratón CD11b </td><td> BioLegend </td><td> 101208 </td><td> clon M1/70; PE acoplado </td></tr><tr><td> anti-ratón CD11c </td><td> BioLegend </td><td> 117310 </td><td> clon N418; APC acoplado </td></tr><tr><td> anti-ratónCD45 </td><td> BioLegend </td><td> 103102 </td><td> clon 30-F11; purificada </td></tr><tr><td> anti-ratón CD19 </td><td> eBioscience </td><td> 12-0193-83 </td><td> eBIO 1D3; PE acoplado </td></tr><tr><td> policlonal de cabra anti-IgG de rata </td><td> BD Pharmingen </td><td> 550767 </td><td> policlonal, PE acoplado </td></tr><tr><td></td><td></td><td></td><td> Anticuerpos acoplados a fluorocromos alternativos pueden ser utilizados, dependiendo del citómetro de flujo y los láseres disponibles. </td></tr></tbody></table>

flow cytometric isolation of primary murine type ii alveolar epithelial cells for functional and molecular studies

A lo largo de los últimos años, la contribución de las células alveolares de tipo II epiteliales (AECII) a diversos aspectos de la regulación inmune en el pulmón se ha reconocido cada vez más. AECII se han mostrado participar en la producción de citoquinas en las vías respiratorias inflamadas y actuar incluso como células presentadoras de antígeno tanto en la infección de células T y la autoinmunidad mediada por 1-8. Por lo tanto, son especialmente interesantes también en contextos clínicos como la hiper-reactividad de las vías respiratorias al exterior y de auto-antígenos, así como infecciones que directa o indirectamente se dirigen AECII. Sin embargo, nuestra comprensión de las funciones inmunológicas detalladas servidos por células alveolares de tipo II epiteliales en el pulmón sano, así como en la inflamación sigue siendo fragmentaria. Muchos estudios sobre la función AECII se realizan a través del ratón o humano líneas de células epiteliales alveolares 9-12. Trabajo con líneas de células sin duda ofrece una serie de beneficios, tales como la disponibilidad de un gran número of células para análisis extensos. Sin embargo, creemos que el uso de AECII murino primaria permite una mejor comprensión de la función de este tipo de células en procesos complejos tales como la infección o inflamación autoinmune. AECII murino primaria se pueden aislar directamente a partir de animales que sufren de tales condiciones respiratorias, lo que significa que han sido objeto de todos los factores extrínsecos adicionales que juegan un papel en el ajuste analizado. Como ejemplo, AECII viable puede ser aislado de ratones por vía intranasal infectadas con virus de influenza A, que se dirige principalmente a estas células para la replicación 13. Es importante destacar que, a través de la infección ex vivo de AECII aislado de ratones sanos, los estudios de las respuestas celulares montados sobre la infección puede ser extendido. El protocolo para el aislamiento de AECII murino primario se basa en la digestión enzimática del pulmón de ratón seguido de etiquetado de la suspensión celular resultante con anticuerpos específicos para CD11c, CD11b, F4/80,CD19, CD45 y CD16/CD32. AECII granulares se identifica entonces como la dispersión de alta sin marcar y lateral (SSC alta) población de células y son separadas por fluorescencia de células activadas por la clasificación 3. En comparación con otros métodos de aislamiento de células epiteliales primarias de los pulmones del ratón, nuestro protocolo de aislamiento de citometría de flujo de AECII por selección negativa produce intacta, altamente viable y puro AECII en tiempo relativamente corto. Además, y en contraste con los métodos convencionales de aislamiento por lavado en batea y el agotamiento de los linfocitos a través de la unión del anticuerpo acoplado-perlas magnéticas 14, 15, de citometría de flujo de clasificación de células permite la discriminación por medio de tamaño de celda y granularidad. Dado que la instrumentación para la clasificación de células por citometría de flujo está disponible, el procedimiento descrito se puede aplicar a costes relativamente bajos. Al lado de anticuerpos estándar y enzimas para la desintegración, pulmón, ausencia de reactivos adicionales tales como magnéticaperlas se requiere. Las células aisladas son adecuadas para una amplia gama de estudios funcionales y moleculares, que incluyen el cultivo in vitro de células T y ensayos de estimulación, así como transcriptoma, proteoma o análisis de secretoma 3, 4.

Al ordenar suspensiones celulares pulmonares aislados de los ratones sanos, la puerta AECII típicamente representan alrededor del 42 ± 10% de todos los eventos. Este porcentaje puede ser notablemente inferior cuando los ratones con enfermedades respiratorias tales como una infección viral se utilizan, como la suspensión de células inicial contendrá una proporción mucho mayor de los linfocitos y otras células inmunitarias reclutados para las vías respiratorias. Para AECII aisladas de los pulmones IAV infectados ...

Watch this Scientific Journal Video about Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies at JoVE.com

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Se describe el aislamiento rápido de células primarias de murino de tipo II alveolares epiteliales (AECII) por citometría de flujo negativo de selección. Estos AECII mostrar una alta viabilidad y pureza, y son adecuados para una amplia gama de estudios funcionales y molecular respecto a su función en condiciones respiratorias tales como enfermedades autoinmunes o infecciosas.

Citometría de flujo El aislamiento de células primarias murino tipo II alveolares epiteliales de Estudios funcionales y moleculares

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

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Immunology and Infection

26.1K vistas.  Helmholtz Centre for Infection Research. Se describe el aislamiento rápido de células primarias de murino de tipo II alveolares epiteliales (AECII) por citometría de flujo negativo de selección. Estos AECII mostrar una alta viabilidad y pureza, y son adecuados para una amplia gama de estudios funcionales y molecular respecto a su función en condiciones respiratorias tales como enfermedades autoinmunes o infecciosas.

Video: Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Análisis por citometría de flujo de células inmunes dentro de aortas murino

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Investigación

Inmunología e Infección

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Análisis del daño epitelial Producido por<em&gt; Entamoeba histolytica</em&gt; Infección

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

El aislamiento, la identificación y purificación de células epiteliales del timo murino

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Aislamiento y la inyección intravenosa de murino médula ósea derivados monocitos

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Aislamiento y análisis de citometría de flujo de células inmunes del cerebro isquémico Ratón

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

El aislamiento y la citometría de flujo análisis de las células mononucleares de sangre periférica Glioma infiltrantes

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

El aislamiento y la citometría de flujo Caracterización de murino Small intestinal linfocitos

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

Aislamiento y Análisis de citometría de flujo, de Human endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Aislamiento y cultivo In Vitro de los macrófagos alveolares murinos y humanos

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

Digestión del hígado murino para el análisis cytometric del flujo de las células endoteliales linfáticas

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...