Este trabajo fue financiado por CIHR y CF subvenciones a Canadá el Dr. Jim Hu y por una beca de doctorado otorgado a Rahul Kushwah por CF Canadá.

1. Sensibilización de los ratones con ovoalbúmina <ol><li> Preparar una solución de OVA (grado V, Sigma, MO) en PBS estéril a una concentración de 1 mg / ml (solución puede ser almacenada a -80 ° C). </li><li> A fin de preparar OVA-Alum mezcla, tomar alumbre en un tubo y añadir solución OVA en una gota a gota mientras vórtex el tubo en una proporción de 1:1. Revuelva la mezcla durante 30 minutos y utilizar de inmediato después de la mezcla. </li><li> Utilizando una jeringa de 1 ml, inyectar 0,2 ml de la mezcla en la cavidad peritoneal de ratón y repetir la inyección de nuevo después de 2 semanas. </li></ol> 2. Desafío intranasal de ratones con ovoalbúmina marcado con FITC <ol><li> 7 días después de la segunda inyección de OVA-Alum, los ratones están listos para ser desafiados por vía intranasal con OVA-FITC. </li><li> Preparar una solución de OVA-FITC (Sigma) utilizando PBS estéril a una concentración de 1 mg / ml y se conserva en alícuotas a -80 ° C. </li><li> Colocar una gasa estéril en la parte inferior de un tubo de 50 ml Falcon y en un h químicaud, añadir 5 ml de Aerrane en la gasa. Con el fin de anestesiar ratones, dirigir el animal en el tubo de aproximadamente 5-10 segundos. </li><li> Sostenga el animal anestesiado en posición vertical y el uso de puntas de pipeta estériles, los Pipetear 100 L de la solución de OVA-FITC en las fosas nasales de los ratones de una forma gota a gota. </li><li> Repita el desafío intranasal con OVA-FITC para los próximos dos días. </li></ol> 3. Preparación de una sola célula de la suspensión de los pulmones y los ganglios linfáticos que drenan <ol><li> El sacrificio de los ratones con la inyección intraperitoneal de Euthanyl. </li><li> Con el fin de reducir la contaminación de macrófagos, que pueden complicar el análisis de CC, lavado pulmonar se lleva a cabo. Para el lavado de los pulmones, tráquea del ratón se expone brevemente y se canuló con un catéter y una jeringa, con helado de PBS con 5 mM EDTA, el tracto respiratorio inferior se lava 3 veces para eliminar las células de los espacios alveolares. </li><li> Perfundir los pulmones con 10 ml de PBS que contenía 10 U / ml Heparen a través del ventrículo derecho del corazón, para eliminar las células de sangre de la vasculatura pulmonar. Realizar las perfusiones hasta que los pulmones se vuelven blancas, lo que indica la eliminación de la mayoría de las células sanguíneas. Esto es especialmente importante para eliminar las células contaminantes de la sangre periférica. </li><li> Diseccionar los pulmones y para quitar los ganglios linfáticos del mediastino (MLN) y poner los ganglios linfáticos en un plato aparte. (Trate de minimizar la exposición de los tejidos a la luz para evitar la extinción de la fluorescencia FITC). </li><li> Coloque los pulmones en una placa de Petri y picar con unas tijeras. Siguiente digerir los pulmones durante 25 minutos a 37 ° C con 250 U / ml de colagenasa D (Roche) y añadir EDTA (10 mM concentración final) para los próximos 5 minutos para detener la actividad de la colagenasa. </li><li> Pase los fragmentos de los pulmones digeridos a través de un colador 100 m celular y la lisis hipotónica para eliminar los eritrocitos. La suspensión de células individuales está listo para su posterior análisis de los países en desarrollo. </li><li> A fin de preparar suspensión de células individuales de la MLN, taliviar el MLN utilizando agujas finas y digerir con colagenasa como se describió anteriormente seguido por el esfuerzo a través de un filtro de células. </li></ol> 4. La tinción de DC marcadores para evaluar la maduración / migración <ol><li> Con el fin de identificar los países en desarrollo en los pulmones, la tinción se lleva a cabo para CD11c y CD11b. Además de evaluar la maduración, la coloración se realiza para CD86 y CD80, que se reguló en los países en desarrollo someterse a la maduración. CD11c + CD11b + OVA-FITC + en las células del MLN se identifican como países en desarrollo pulmonar que migran desde los pulmones al MLN en respuesta al reto de las vías respiratorias OVA. </li><li> Suspender las células a una concentración de 10 x 10 6 células / ml en tampón FACS (PBS con FBS al 1% y EDTA 1 mM) y alícuota 150 l de suspensión de células / tubo para la tinción FACS. Para la identificación de los países en desarrollo pulmonar, las manchas se necesitan los siguientes: el control no teñida (células de pulmón de los ratones no expuestos a la OVA-FITC), OVA-FITC, control de CD11c sola, CD11b solo mando, CD11b+ CD11c + doble control, CD11b + OVA-FITC + doble control, CD11c + OVA-FITC + doble control, MHC II de control único, el control de un solo CD86, CD80 única de control y las muestras teñidas con OVA-FITC + CD11c + CD11b + + MHCII , OVA-FITC + CD11c + CD86 + CD11b + y OVA-FITC + CD11c + CD80 + CD11b +. </li><li> Para evaluar la migración de CC, realizar recuentos absolutos de células de la suspensión de células individuales del MLN y, posteriormente, preparar los tubos siguientes: control de células sin teñir (MLN de los ratones no expuestos a la OVA-FITC), el control de OVA-FITC, CD11c + control único, CD11b + doble control, el control de CD11b + OVA-FITC + doble, control de CD11c + OVA-FITC + doble y CD11c + CD11b + OVA-FITC + muestras. </li></ol> 5. Los resultados representativos Los puntos de tiempo requeridos para la sensibilización intraperitoneal para inducir inflamación de las vías alérgica es importante y debe ser llevado a cabo como se representa en la Figura 1. Tras la sensibilización intraperitoneal y OVA desafío vías respiratorias, para confirmar la inducción de la inflamación de las vías alérgica, algunos ratones pueden ser sacrificados y los análisis histológicos puede llevarse a cabo en las secciones de pulmón, como se muestra en la Figura 2. La presencia de células inflamatorias puede ser confirmado por Hematoxilina y Eosina (Figura 2A) y la presencia de la producción de moco puede ser evaluada por ácido periódico de Schiff-tinción (Figura 2B). En total, esto confirma la inducción de la inflamación de las vías respiratorias alérgicas después de OVA desafío de OVA-ratones sensibilizados a 8. En contraste, las secciones de pulmón de solución salina ratones tratados se espera que sea libre de cualquier inflamación junto con ausencia de cualquier producción de moco. Por otra parte, OVA desafío siguiente, pulmonar unde los países en desarrollo<html> RGO maduración y posterior migración a los ganglios linfáticos de drenaje. El análisis de células CD11c + en los ganglios linfáticos del mediastino (MLN) de los ratones sensibilizados y desafiados con OVA se espera que muestre una mayor proporción de células CD11c + en comparación con los ratones tratados con solución salina (Figura 3). Además, el análisis del recuento de linfocitos absoluto de CD11c + CD11b + OVA-FITC + en las células en el MLN de los ratones OVA-sensibilizado y desafiado, se espera que muestre un valor significativamente mayor (es decir, varias veces superior) cuenta que los homólogos de los ratones tratados con solución salina ( Figura 3B). <img alt="Figura 1" src="/files/ftp_upload/4014/4014fig1.jpg" /> Figura 1. Protocolo experimental para la inducción de la ovoalbúmina (OVA) la inflamación inducida por las vías respiratorias alérgicas en ratones junto con el reto de las vías respiratorias con marcado con FITC ovoalbúmina (OVA-FITC). fig2.jpg &quot;/&gt; Sensibilización Figura 2. OVA seguido por las vías respiratorias reto OVA conduce a la inducción de la inflamación de las vías alérgica identificadas por tinción de hematoxilina y eosina de secciones de pulmón se muestran en (A) y también conduce a la producción de moco en las vías respiratorias como identificadas por tinción periódico de Schiff ácido de pulmón secciones como se muestra en (B). <img alt="Figura 3" src="/files/ftp_upload/4014/4014fig3.jpg" /> Figura 3. OVA-FITC desafío induce la migración de CC de los pulmones a los ganglios linfáticos del mediastino (MLN). (A) parcelas de citometría de flujo que representa la proporción de células CD11c + en el MLN de control o los ratones sensibilizados con OVA. (B) Los recuentos absolutos de CD11c + células CD11b + + OVA-FITC en el MLN de los ratones solución salina u OVA sensibilizado. * P &lt;0,05. <a href="http://www.jove.com/files/ftp_upload/4014/4014fig3large.jpg" target="_blank">Haga clic aquí para ver más grande la figura</a> .

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	<li>Kushwah, R., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+dendritic+cells+in+the+induction+of+regulatory+T+cells.">Role of dendritic cells in the induction of regulatory T cells.</a> Cell Biosci. 20, (2011).</li><li>Kushwah, R., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexity+of+dendritic+cell+subsets+and+their+function+in+the+host+immune+system.">Complexity of dendritic cell subsets and their function in the host immune system.</a> Immunology. 133, 409-419 (2011).</li><li>Lambrecht, B. N., Hammad, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+our+breath+away:+dendritic+cells+in+the+pathogenesis+of+asthma.">Taking our breath away: dendritic cells in the pathogenesis of asthma.</a> Nat Rev Immunol. 3, 994-1003 (2003).</li><li>Hammad, H., Lambrecht, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+epithelial+cells:+linking+innate+and+adaptive+immunity+in+asthma.">Dendritic cells and epithelial cells: linking innate and adaptive immunity in asthma.</a> Nat. Rev. Immunol. 8, 193-204 (2008).</li><li>Lloyd, C. M., Murdoch, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerizing+allergic+responses+in+the+lung.">Tolerizing allergic responses in the lung.</a> Mucosal. Immunol. 3, 334-344 (2010).</li><li>Herz, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+of+murine+animal+models+to+study+the+development+of+allergic+bronchial+asthma.">The relevance of murine animal models to study the development of allergic bronchial asthma.</a> Immunol. Cell Biol. 74, 209-217 (1038).</li><li>Herz, U., Lumpp, U., Daser, A., Gelfand, E. W., Renz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+animal+models+to+study+the+central+role+of+T+cells+in+immediate-type+hypersensitivity+responses.">Murine animal models to study the central role of T cells in immediate-type hypersensitivity responses.</a> Adv. Exp. Med. Biol. 409, 25-32 (1996).</li><li>Nakae, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF+can+contribute+to+multiple+features+of+ovalbumin-induced+allergic+inflammation+of+the+airways+in+mice.">TNF can contribute to multiple features of ovalbumin-induced allergic inflammation of the airways in mice.</a> J. Allergy Clin. Immunol. 119, 680-686 (2007).</li><li>Guth, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+environment+determines+unique+phenotype+of+alveolar+macrophages.">Lung environment determines unique phenotype of alveolar macrophages.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 296, L936-L946 (2009).</li><li>Kushwah, R., Cao, H., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+pulmonary+T+cell+response+to+helper-dependent+adenoviral+vectors+following+intranasal+delivery.">Characterization of pulmonary T cell response to helper-dependent adenoviral vectors following intranasal delivery.</a> J. Immunol. 180, 4098-4108 (2008).</li><li>Kushwah, R., Oliver, J. R., Zhang, J., Siminovitch, K. A., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+dendritic+cells+induce+tolerance+in+mice+through+suppression+of+dendritic+cell+maturation+and+induction+of+antigen-specific+regulatory+T+cells.">Apoptotic dendritic cells induce tolerance in mice through suppression of dendritic cell maturation and induction of antigen-specific regulatory T cells.</a> J. Immunol. 183, 7104-7118 (2009).</li></ol>

No hay conflictos de interés declarado.

El método que aquí se presenta ofrece un enfoque basado en la citometría de flujo para el análisis de los países en desarrollo pulmonar, con base en la entrega de OVA-FITC para el desafío de las vías respiratorias. Esto permite el control selectivo de los países en desarrollo pulmonar, que ocupan un OVA-FITC y por lo tanto, las poblaciones de CC, que se controlan son efectivamente los que participan en la respuesta inmune vías respiratorias durante el curso de la inflamación inducida por OVA las vías respirat...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Ovoalbúmina </td><td> Sigma-Aldrich </td><td> A5503 </td></tr><tr><td> Alumbre </td><td> Thermo Scientific </td><td> 77161 </td></tr><tr><td> OVA-FITC </td><td> Sigma-Aldrich </td><td> A9771 </td></tr><tr><td> Colagenasa D </td><td> Roche </td><td> 11088858001 </td></tr><tr><td> Anticuerpos </td><td> e-Bioscience </td><td></td></tr></tbody></table>

analysis of pulmonary dendritic cell maturation and migration during allergic airway inflammation

Las células dendríticas (DC) son los principales actores implicados en la iniciación de la respuesta inmune adaptativa mediante la activación de antígenos específicos de células T. Los países en desarrollo están presentes en los tejidos periféricos en estado estacionario, sin embargo, en respuesta a la estimulación antigénica, los países en desarrollo ocupan el antígeno y rápidamente migran a los ganglios linfáticos que drenan en el que inician la respuesta de células T contra el antígeno de 1,2. Además, los países en desarrollo también desempeñan un papel clave en la iniciación de la respuesta inmunitaria autoinmune, así como alérgica 3. Los países en desarrollo desempeñan un papel esencial tanto en la iniciación de la respuesta inmune y la inducción de la tolerancia en el ámbito del medio ambiente de pulmón 4. Entorno de pulmón es en gran parte tolerogénico, debido a la exposición a la amplia gama de antígenos ambientales 5. Sin embargo, en algunos individuos hay una ruptura en la tolerancia, lo que conduce a la inducción de la alergia y el asma. En este estudio, se describe una estrategia, que se puede utilizar para controlar las vías respiratorias maduración DC y MIGración en respuesta al antígeno utilizado para la sensibilización. La medición de las vías respiratorias maduración de las DC y la migración permite la evaluación de la cinética de la respuesta inmune durante la inflamación las vías respiratorias alérgicas y también ayuda a comprender la magnitud de la posterior respuesta inmune junto con los mecanismos subyacentes. Nuestra estrategia se basa en la utilización de ovoalbúmina como agente sensibilizante. Ovoalbúmina inducida por el asma alérgica es un modelo ampliamente utilizado para reproducir la eosinofilia las vías respiratorias, inflamación pulmonar y niveles elevados de IgE se encuentran en el asma 6,7. Después de la sensibilización, los ratones son desafiados por la administración intranasal de ovoalbúmina marcado con FITC, lo que permite el etiquetado específico de los países en desarrollo las vías respiratorias, que la captación de ovoalbúmina. A continuación, utilizando varios marcadores específicos de CC, podemos evaluar la maduración de estos países en desarrollo y también se puede evaluar su migración a los ganglios linfáticos de drenaje mediante el empleo de la citometría de flujo.

Watch this Scientific Journal Video about Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation at JoVE.com

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Se describe una estrategia para controlar la maduración y la migración de las células dendríticas pulmonares en respuesta a la ovoalbúmina en la configuración de las vías respiratorias inflamación inducida por ovalbúmina alérgica. Esta estrategia se puede modificar para evaluar la migración de las células dendríticas pulmonares en la configuración de la infección.

El análisis de la maduración pulmonar de células dendríticas y migración durante la inflamación de las vías alérgica

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

analysis-pulmonary-dendritic-cell-maturation-migration-during

Research

JoVE Journal

Immunology and Infection

14.9K vistas.  McMaster University, Hamilton. Se describe una estrategia para controlar la maduración y la migración de las células dendríticas pulmonares en respuesta a la ovoalbúmina en la configuración de las vías respiratorias inflamación inducida por ovalbúmina alérgica. Esta estrategia se puede modificar para evaluar la migración de las células dendríticas pulmonares en la configuración de la infección.

Video: Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Aislamiento de linfocitos T humanos, cultura y análisis de la migración In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Investigación

Inmunología e Infección

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Protocolo optimizado para la transfección eficiente de células dendríticas, sin maduración de las células

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

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 ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

De las Migraciones células dendríticas mediante resonancia 19F / 1H Magnetic

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

In vivo método de imagen para distinguir la inflamación aguda y crónica

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

El aislamiento, caracterización y examen funcional de la red celular gingival Inmune

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

Administración intra-traqueal de<em&gt; Haemophilus influenzae</em&gt; En modelos de ratón para estudiar las vías respiratorias inflamación

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

Un ensayo basado en CFSE para estudiar la migración de la piel murina de células dendríticas en los nodos linfáticos de drenaje durante la infección con<em&gt; Mycobacterium bovis</em&gt; Bacilo de Calmette-Guérin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

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 ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Análisis de cortante inducida por el flujo de migración de las células B de zona Marginal de Murine In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...