Los autores desean agradecer a Marilyn Dietrich en el Centro de Citometría de Flujo LSU central por su ayuda en la clasificación de células, y Mottram Pedro por su ayuda con las microfotografías. Este trabajo fue financiado por el Flight Attendant Medical Research Institute, el Premio de Investigación LSU-programa competitivo, y las subvenciones del NIH / NIAID P20 RR020159 y R03AI081171.

1. Perfusión pulmonar y la suspensión de células individuales <ol><li> La eutanasia a los ratones con una inyección intraperitoneal de ketamina / xilazina mezcla anestésica (en mg / ratón ketamina 1.8, xilazina 0,19) y desangrado por la vena femoral </li><li> Exponer la cavidad torácica por el corte y retraiga la piel exterior del peritoneo. Procederá a la apertura del diafragma por el corte de la caja torácica para exponer el corazón y los pulmones. </li><li> Perfusión de los pulmones con suavidad utilizando una jeringa de 5 ml llena de EDTA-HBSS (1 mM EDTA en magnesio libre de calcio / HBSS). Conecte una aguja G 25 e insertar la aguja en el ventrículo derecho. Para evitar fugas durante la inyección, seguro que el tejido de miocardio alrededor de la aguja por el uso de fórceps. Inyectar suavemente la solución, manteniendo una presión constante. Perfusión precisa dará lugar a la inflación de pulmón y un cambio de color de rosa / blanco. </li><li> Quitar ganglios linfáticos de drenaje para descartar cualquier posible contaminación de este tejido. </li><li> Recogertejido pulmonar, la transferencia de una placa de Petri en el hielo, y cortar en pedazos pequeños, usando una cuchilla de afeitar. </li><li> La transferencia de tejido a tierra en un tubo de gentleMACS C con 5 ml / pulmón de la solución de colagenasa digestión (colagenasa tipo 1A 0,5 mg / ml, más de tipo IV pancreática bovina DNasa 20 mg / ml en HBSS que contiene 5% de SFB, 100 U / ml penicilina y 100 mg / ml estreptomicina). </li><li> Utilice el disociador gentleMACS para obtener una suspensión de células individuales. Elección del programa: m_lung_01. Incubar la muestra a 37 ° C durante 30 minutos. Agite el tubo cada 5 minutos para volver a suspender fragmentos de tejido. Proceder con m_lung_02 programa. </li><li> Transferir la muestra a un tubo cónico de 15 ml y centrifugar durante 10 minutos a 335 x g a 4 ° C. Después de este paso, tenga todos los reactivos y centrifugados a 4 ° C para evitar una reducción de la viabilidad celular. </li><li> Desechar el sobrenadante y el resto de lisar los glóbulos rojos mediante la adición de 2 ml de tampón de lisis ACK durante 1 minuto a temperatura ambiente. Lavar las células con 13ml de BSA frío PBS/0.5% y centrifugar durante 10 minutos a 335 x g a 4 ° C. </li><li> Deseche las células sobrenadante, el total de resuspender en 5 ml de BSA frío PBS/0.5% y pasa a fondo una malla de 100 micras de nylon. </li><li> Determinar el número total de células mediante el uso de tinte de exclusión con azul tripán. </li></ol> 2. Aislamiento magnético y el enriquecimiento de las células CD11c + <ol><li> Centrifugar la suspensión de células individuales de 10 minutos a 200 x g a 4 º C y desechar el sobrenadante. </li><li> Resuspender el sedimento celular en 400 l de tampón de separación (PBS, 0,5% de BSA y 2 mM EDTA) por 10 8 células totales. Bloquear Fc-mediada por anticuerpos no específicos de enlace mediante anti-CD16/CD32 anticuerpos (0.5μg / 10 6 células) durante 30 minutos a 4 ° C. </li><li> Lavar las células con 10 ml de tampón de separación frío y centrifugar durante 10 minutos a 200 x g a 4 ° C. Resuspender las células en 400 l de tampón de separación. </li><li> Añadir 100 ml de MicroBeads CD11c por 10 8 </apoyo células&gt; total. Mezclar bien e incubar durante 15 minutos en el refrigerador (2-8 ° C). </li><li> Lavar las células con 10 ml de tampón de separación frío y centrifugar durante 10 minutos a 200 x g a 4 º C y desechar el sobrenadante. </li><li> Resuspender el botón celular en 3 ml de tampón de separación de frío. </li><li> Realizar la separación magnética utilizando el separador autoMACS Pro TM seleccionando el posseld programa. Recoger la fracción positiva (0,5 ml). </li></ol> 3. Células dendríticas convencionales (CDC) de aislamiento <ol><li> Incubar enriquecido CD11c suspensión de células positivas a anti-CD11c-PE Cy7 y anti-IA/IE (MHC-II)-FITC anticuerpos durante 30 minutos a 4 ° C. Concentración de anticuerpos optimizado es de 0,5 mg / 10 6 de las células. </li><li> Lavar las células con BSA frío PBS/0.5% y centrifugar durante 10 minutos a 335 x g a 4 ° C. Resuspender las células en 0,5 ml de PBS/0.5% de BSA y pasar a través de una malla de 40 micras de nylon. </li><li> Proceder a laordenar a las células mediante el uso de la FACS Aria clasificador de células en el modo de pureza. Añadir 100 ml de PBS/0.5% de BSA para los tubos de recolección con el fin de prevenir el daño de las células purificadas. Mantener a las células a una temperatura refrigerada durante todo el proceso de separación celular. </li></ol> 4. Protocolo alternativo para la obtención de una suspensión de células individuales y enriquecimiento de las células CD11c + <ol><li> En sustitución del disociador gentleMACS, el tejido pulmonar a tierra desde el paso 1.5 se puede transferir a un tubo cónico de 15 ml que contiene 5 ml de solución de colagenasa por los pulmones y se incuba durante 1 hora a 37 ° C. Vortex células cada 15 minutos con el fin de volver a suspender fragmentos de tejido. Destruir el tejido de pasar la muestra a 6-8 veces de entrada y salida a través de una jeringa de 3 ml conectada a una aguja de 20 G. Evitar hacer burbujas durante el proceso, de lo contrario la viabilidad celular se verá comprometida. Una vez que la suspensión de células individuales se obtiene, continúe con el paso 1.8. </li><li> En sustitución de la separación AutoMACSPro TMtor, aislamiento magnético para el enriquecimiento de las células CD11c, también se puede realizar al aprobar la suspensión de células de forma manual a través de dos columnas de MACS. La selección de las columnas necesarias variarán en función del tamaño de la muestra. </li></ol> 5. Los resultados representativos: Pulmonar CDC se identifican como CD11c hi / MHC-II la población de células de alta. Como se muestra en la fig. 1, los CDC representan un porcentaje menor del 1,4% en comparación con algunos otros tejidos como el bazo (4,8%). Sin embargo, en contraste con el bazo, el pulmón de células CD11c positivo expresan diferentes cantidades de MHC-II, incluyendo una población de células diferentes que la de los CDC. Después de la preparación de una sola célula, la producción de células de pulmón de células total era de aproximadamente 3,0 a 3,8 x 10 7 células / pulmonar con una viabilidad de 60 a 70% de los cuales un poco más del 1% representado subconjunto CDC. Este porcentaje se incrementó después de que el aislamiento CD11c magnética donde el total del pulmón CDC se incrementó alrededor de 10 tIME (&gt; 16%) de la preparación original (Fig. 2A). Sin embargo, las células que expresan CD11c bajas cantidades de MHC-II (CD11c hi / lo MHC-II, los macrófagos) representa una alta contaminación de la población de células (&gt; 70%), que se mezcló con el subconjunto de pulmón CDC. Como se muestra en la fig. 2B, las células fueron eliminados tras la etapa de clasificación de células, cuando la pureza CDC alcanzó&gt; 96%. El rendimiento normal de los CDC después de todo el procedimiento fue de 5 x 10 4 células / pulmón. Microfotografías muestran las características morfológicas de los CDC como células redondeadas con las dendritas típicas (panel superior). Por otro lado, CD11c hi / lo MHC-II representa un subconjunto morfológicamente diferentes que muestra protuberancias celulares que son típicas de los macrófagos (MΦ, panel inferior) 12. <img alt="Figura 1" src="/files/ftp_upload/3563/3563fig1.jpg" /> Figura 1. Diferencial DC frecuencia en el pulmón y el bazo del ratón. Un pulmóntejido º del bazo de ratones C57BL / 6 fueron recogidos y tratados con colagenasa. Después de la digestión colagenasa, las células fueron teñidas con anti-ratón CD11c-PE-IA Cy7 y anti-ratón / IE-FITC. Representante de las parcelas de citometría de flujo muestran los porcentajes de los CDC (CD11c de alta / alta MHC-II) en el pulmón y el bazo. <img alt="Figura 2" src="/files/ftp_upload/3563/3563fig2.jpg" /> Figura 2. Aislamiento de los CDC de pulmón de ratón. Suspensión de ratón de pulmón de células individuales fue marcado con anti-CD11c microesferas y pasa a través de un separador celular automático. A) Un gráfico muestra los porcentajes de representación de los CDC (CD11c de alta / alta MHC-II) y macrófagos (MΦ, CD11c hi / lo MHC-II) la población después del enriquecimiento CD11c. Tinción más de la fracción enriquecida seguido utilizando anti-ratón CD11c-PE-IA Cy7 y anti-ratón / IE-FITC. Doble células positivas fueron ordenados y analizados por citometría de flujo. Aidentificar su morfología, las células fueron cytospun y se tiñeron con una versión modificada de Wright-Giemsa tinción. B) Representante parcelas muestran los porcentajes de los CDC y MΦ después de la separación de células. Microfotografías muestran una imagen representativa de pulmón CDC y MΦ pulmón. Barra de escala = 20 micras.

<ol>
	<li>Pulendran, B., Palucka, K., Banchereau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+pathogens+and+tuning+immune+responses.">Sensing pathogens and tuning immune responses.</a> Science. 293, 253-256 (2001).</li><li>Banchereau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunobiology+of+dendritic+cells.">Immunobiology of dendritic cells.</a> Annual Review of Immunology. 18, 767-811 (2000).</li><li>Manicassamy, S., Pulendran, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+control+of+tolerogenic+responses.">Dendritic cell control of tolerogenic responses.</a> Immunol. Rev. 241, 206-227 (2011).</li><li>Steinman, R. M., Cohn, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+cell+type+in+peripheral+lymphoid+organs+of+mice.+I.+Morphology,+quantitation,+tissue+distribution.">Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution.</a> J. Exp. Med. 137, 1142-1162 (1973).</li><li>Asselin-Paturel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+type+I+IFN-producing+cells+are+immature+APCs+with+plasmacytoid+morphology.">Mouse type I IFN-producing cells are immature APCs with plasmacytoid morphology.</a> Nat. Immunol. 2, 1144-1150 (2001).</li><li>Chan, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-producing+killer+dendritic+cells+provide+a+link+between+innate+and+adaptive+immunity.">Interferon-producing killer dendritic cells provide a link between innate and adaptive immunity.</a> Nat. Med. 12, 207-213 (2006).</li><li>Taieb, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+dendritic+cell+subset+involved+in+tumor+immunosurveillance.">A novel dendritic cell subset involved in tumor immunosurveillance.</a> Nat. Med. 12, 214-219 (2006).</li><li>Guerrero-Plata, A., Kolli, D., Hong, C., Casola, A., Garofalo, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subversion+of+pulmonary+dendritic+cell+function+by+paramyxovirus+infections.">Subversion of pulmonary dendritic cell function by paramyxovirus infections.</a> Journal of Immunology. 182, 3072-3083 (2009).</li><li>Larson, R. S., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+leukocyte+integrins.">Structure and function of leukocyte integrins.</a> Immunol. Rev. 114, 181-217 (1990).</li><li>Sung, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+major+lung+CD103+(alphaE)-beta7+integrin-positive+epithelial+dendritic+cell+population+expressing+Langerin+and+tight+junction+proteins.">A major lung CD103 (alphaE)-beta7 integrin-positive epithelial dendritic cell population expressing Langerin and tight junction proteins.</a> J. Immunol. 176, 2161-2172 (2006).</li><li>Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+CD11c++MHC+class+II-+precursors+generate+lung+dendritic+cells+during+respiratory+viral+infection,+but+are+depleted+in+the+process.">Local CD11c+ MHC class II- precursors generate lung dendritic cells during respiratory viral infection, but are depleted in the process.</a> J. Immunol. 177, 2536-2542 (2006).</li><li>Bhatia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+host+defense+against+Aspergillus+fumigatus+involves+alveolar+macrophages+with+a+predominance+of+alternatively+activated+phenotype.">Rapid host defense against Aspergillus fumigatus involves alveolar macrophages with a predominance of alternatively activated phenotype.</a> PLoS One. 6, e15943-e15943 (2011).</li><li>Shao, Z., Makinde, T. O., McGee, H. S., Wang, X., Agrawal, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fms-like+tyrosine+kinase+3+ligand+regulates+migratory+pattern+and+antigen+uptake+of+lung+dendritic+cell+subsets+in+a+murine+model+of+allergic+airway+inflammation.">Fms-like tyrosine kinase 3 ligand regulates migratory pattern and antigen uptake of lung dendritic cell subsets in a murine model of allergic airway inflammation.</a> J. Immunol. 183, 7531-75381 (2009).</li><li>Hao, X., Kim, T. S., Braciale, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+response+of+respiratory+dendritic+cell+subsets+to+influenza+virus+infection.">Differential response of respiratory dendritic cell subsets to influenza virus infection.</a> J. Virol. 82, 4908-4919 (2008).</li></ol>

Los autores no tienen nada que revelar.

El aislamiento de DC pulmonar del ratón es una técnica importante para el estudio de una amplia gama de estímulos respiratorios. El proceso de obtención de estas células incluye los pasos críticos que impiden la pérdida de las células, así como la viabilidad celular y la pureza. Perfusión del pulmón antes de la recogida ayudará a eliminar las células periféricas, así como reducir los eritrocitos contaminantes. El uso de la disociación puede ser automatizado advanteogus cuando un gran número de los pulmo...

<table width="200" border="1"><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Buffer de lisis ACK </td><td> Invitrogen </td><td> D6-0005DG </td><td></td></tr><tr><td> Anti-ratón CD11c (HL3) </td><td> BD Pharmingen </td><td> 5580979 </td><td> PE-conjugado Cy7 </td></tr><tr><td> Anti-ratón de IA / IE (269) </td><td> BD-Pharmingen </td><td> 553623 </td><td> Conjugado con FITC </td></tr><tr><td> Collangenase tipo 1A </td><td> Sigma </td><td> 9891-500MG </td><td></td></tr><tr><td> Filtros de células </td><td> BD Falcon </td><td> 352340, 352360 </td><td></td></tr><tr><td> CD11c (N418) microesferas </td><td> Miltenyi </td><td> 130-052-001 </td><td></td></tr><tr><td> DNasa I </td><td> Sigma </td><td> D5025-150KU </td><td></td></tr><tr><td> Solución salina equilibrada de Hank </td><td> Invitrogen </td><td> 14170 </td><td></td></tr><tr><td> Solución tampón Hepes </td><td> Invitrogen </td><td> 15630 </td><td></td></tr><tr><td> Placas de Petri de 60 mm </td><td> BD Falcon </td><td> 351016 </td><td></td></tr><tr><td> GentleMACS ™ C tubos </td><td> Miltenyi </td><td> 130-093-237 </td><td></td></tr><tr><td> MACS disociador suave </td><td> Miltentyi </td><td> 130-093-235 </td><td></td></tr><tr><td> AutoMACS-Pro ™ </td><td> Miltenyi </td><td> 130-092-545 </td><td></td></tr><tr><td> FASC Aria </td><td> BD </td><td></td><td></td></tr></table>

isolation of mouse lung dendritic cells

Las células pulmonares dendríticas (DC), juegan un papel fundamental en la detección de 1,2 patógenos invasores, así como en el control de las respuestas tolerogénicas 3 en el tracto respiratorio. Al menos tres subgrupos principales de las células dendríticas del pulmón se han descrito en ratones: CC convencionales (CDC) 4, plasmocitoide DC (PDC) 5 y el asesino DC IFN-producción (IKDC) 6,7. El subconjunto de los CDC es el subconjunto DC más destacados en el pulmón 8. El marcador común conocida para identificar subgrupos de DC CD11c es, un tipo que la integrina transmembrana (β2) que también se expresa en monocitos, macrófagos, neutrófilos y algunas células B 9. En algunos tejidos, con CD11c como marcador para identificar ratón DC es válido, como en el bazo, donde la mayoría de las células CD11c + representan el subconjunto de los CDC, que expresa los altos niveles del complejo mayor de histocompatibilidad de clase II (MHC-II). Sin embargo, el pulmón es un tejido más heterogénea, donde sesubconjuntos lado DC, hay un alto porcentaje de una población de células distintas que expresa los altos niveles de CD11c pelea niveles bajos de MHC-II. Con base en su caracterización y sobre todo en su expresión de F4/80, un marcador de macrófagos del bazo, el CD11c alta MHC-II pulmonar he población celular ha sido identificado como macrófagos pulmonares 10 y más recientemente, como un posible precursor de DC 11. En contraste con el ratón PDC, el estudio de la función específica de los CDC en la respuesta inmune pulmonar ha sido limitado debido a la falta de un marcador específico que podría ayudar en el aislamiento de estas células. Por lo tanto, en este trabajo se describe un procedimiento para aislar altamente purificada de pulmón de ratón CDC. El aislamiento de los subconjuntos pulmonar DC representa una herramienta muy útil para obtener información sobre la función de estas células en respuesta a patógenos respiratorios, así como los factores ambientales que pueden desencadenar la respuesta inmune del huésped en el pulmón.

Watch this Scientific Journal Video about Isolation of Mouse Lung Dendritic Cells at JoVE.com

Isolation of Mouse Lung Dendritic Cells

Una preparación altamente purificada de células dendríticas del pulmón del ratón se describe. Especial énfasis se da al aislamiento de subgrupo de células dendríticas convencionales.

Aislamiento de células dendríticas del pulmón de ratón

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Research

JoVE Journal

Immunology and Infection

28.9K vistas.  Louisiana State University. Una preparación altamente purificada de células dendríticas del pulmón del ratón se describe. Especial énfasis se da al aislamiento de subgrupo de células dendríticas convencionales.

Video: Isolation of Mouse Lung Dendritic Cells

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

Aislamiento de células de ratón cavidad peritoneal

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Investigación

Inmunología e Infección

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Aislamiento y caracterización de las células dendríticas y los macrófagos del intestino delgado de ratón

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies 2.
Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production.
DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 &deg;C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine 3. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4 - 20 x 106 cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum specifications before they are injected into patients.

The most commonly used method for generating large numbers of autologous dendritic cells (DCs) for use in tumor immunotherapy is described. The method uses IL-4 and GM-CSF to differentiate DCs from monocytes. The immature DCs are stimulated to mature and then loaded with antigens before they are injected back into the patient.

Preparación de las células dendríticas maduras cargadas con antígeno del tumor para inmunoterapia

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

Aislamiento de células dendríticas mieloides y células epiteliales de timo humano

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Un método de rendimiento eficiente y de alta para el aislamiento de ratón subconjuntos de células dendríticas

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Clasificación de células activadas por fluorescencia para la purificación de células dendríticas plasmocitoide de la médula ósea de ratones

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Generación de células dendríticas derivadas de monocitos humanos de sangre entera

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Legionella pneumophila exterior vesículas de membrana: Aislamiento y análisis de su potencial pro-inflamatoria en los macrófagos

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

Las células dendríticas humanas de pulmón: distribución espacial y fenotípica de identificación en endobronquial biopsias mediante inmunohistoquímica y citometría de flujo

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Precisión de corte pulmón de ratón Rebanadas para visualizar las células en vivo pulmonar dendríticas

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...