Ce travail a été financé par des subventions des IRSC et des FC au Canada au Dr Jim Hu et par une bourse de doctorat décerné à Rahul Kushwah par CF Canada.

1. Sensibilisation des souris avec de l&#39;ovalbumine <ol><li> Préparer une solution d&#39;OVA (grade V; Sigma, MO) dans du PBS stérile à une concentration de 1 mg / ml (solution peut être conservée à -80 ° C). </li><li> Afin de préparer OVA-Alum mélange, prendre alun dans un tube et ajouter la solution d&#39;OVA dans un goutte à goutte tout en vortex le tube à un ratio de 1:1. Remuer le mélange pendant 30 minutes et utiliser tout de suite après le mélange. </li><li> En utilisant une seringue de 1 ml, injecter 0,2 ml du mélange dans la cavité péritonéale de la souris et répétez l&#39;injection à nouveau au bout de 2 semaines. </li></ol> 2. Défi intranasale de souris avec de l&#39;ovalbumine étiqueté FITC <ol><li> 7 jours après la deuxième injection de OVA-Alum, les souris sont prêts à être contestée par voie intranasale avec OVA-FITC. </li><li> Préparer une solution d&#39;OVA-FITC (Sigma) à l&#39;aide du PBS stérile à une concentration de 1 mg / ml et conserver en aliquots à -80 ° C. </li><li> Placez une gaze stérile au fond d&#39;un tube Falcon de 50 ml et dans un h chimiqueood, ajoutez 5 ml de Aerrane sur la gaze. Afin d&#39;anesthésier les souris, diriger l&#39;animal dans le tube pendant quelques secondes environ 5-10. </li><li> Maintenez l&#39;animal anesthésié dans une position verticale et en utilisant des pointes de pipette stériles, pipette 100 ul de la solution d&#39;OVA-FITC sur les narines de souris dans un mode goutte à goutte. </li><li> Répétez défi intranasale avec de l&#39;OVA-FITC pour les deux prochains jours. </li></ol> 3. Préparation de la simple cellule de suspension à partir des poumons et les ganglions lymphatiques drainant <ol><li> Sacrifiez les souris en utilisant une injection intrapéritonéale de Euthanyl. </li><li> Afin de réduire la contamination des macrophages, ce qui peut compliquer l&#39;analyse DC, lavage du poumon est réalisée. Pour un lavage des poumons, trachées de souris sont brièvement exposés et une canule avec un cathéter et d&#39;une seringue; en utilisant PBS glacé avec 5 mM d&#39;EDTA, du tractus respiratoire inférieur est rincé 3 fois pour enlever les cellules à partir des espaces alvéolaires. </li><li> Perfuser les poumons avec 10 ml de PBS contenant 10 U / ml hepardans le ventricule droit par l&#39;intermédiaire du cœur, pour éliminer les cellules sanguines du système vasculaire des poumons. Effectuez les perfusions jusqu&#39;à ce que les poumons deviennent blancs, indicative de la suppression de la plupart des cellules sanguines. Ceci est particulièrement important pour éliminer les cellules contaminantes du sang périphérique. </li><li> Disséquer les poumons et enlever les ganglions lymphatiques médiastinaux (MLN) et placez les ganglions lymphatiques dans un plat à part. (Essayez de minimiser l&#39;exposition des tissus à la lumière pour éviter la trempe de FITC fluorescence.) </li><li> Placez les poumons sur une boîte de Pétri et émincer l&#39;aide de ciseaux. Suivant digérer les poumons pendant 25 minutes à 37 ° C en utilisant 250 U / ml de collagénase D (Roche) et ajouter de l&#39;EDTA (10 mM de concentration finale) pour les 5 prochaines minutes pour arrêter l&#39;activité de la collagénase. </li><li> Passez les fragments de poumons digérés à travers une passoire 100 um cellulaire et d&#39;effectuer une lyse hypotonique à éliminer les érythrocytes. La suspension cellulaire unique est prêt pour une analyse plus poussée des pays en développement. </li><li> Afin de préparer la suspension cellulaire unique à partir du MLN, tfaciliter la MLN l&#39;aide d&#39;aiguilles fines et digérer avec de la collagénase comme décrit ci-dessus suivie par la tension à travers un tamis cellulaire. </li></ol> 4. Coloration pour DC Marqueurs pour évaluer la maturation / migration <ol><li> Afin d&#39;identifier les pays en développement dans les poumons, la coloration est effectuée pour CD11c et CD11b. En outre d&#39;évaluer la maturation, la coloration est effectuée pour CD86 et CD80, qui sont régulés à la hausse comme PED subissent une maturation. CD11c + CD11b cellules + + OVA-FITC dans le MLN sont identifiés comme les PED pulmonaires migrateurs à partir des poumons vers le MLN en réponse au défi des voies respiratoires OVA. </li><li> Suspendre les cellules à une concentration de 10 x 10 6 cellules / ml dans tampon FACS (PBS avec 1% de FBS et 1 mM EDTA) et aliquote 150 pi de suspension cellulaire / tube pour la coloration FACS. Pour l&#39;identification des pays en développement du poumon, taches suivantes sont nécessaires: le contrôle non marqué (cellules pulmonaires de souris ne sont pas exposés à l&#39;OVA-FITC), OVA-FITC de contrôle, CD11c de contrôle unique, CD11b de contrôle unique, CD11b+ CD11c + double contrôle, CD11b + OVA-FITC + double contrôle, CD11c + OVA-FITC + double contrôle, CMH II de contrôle unique, CD86 de contrôle unique, CD80 de contrôle unique et des échantillons colorés pour OVA-FITC + CD11c + CD11b + + MHCII , OVA-FITC + CD11c + CD86 + CD11b + et OVA-FITC + CD11c + CD80 + CD11b +. </li><li> À évaluer la migration DC, la numération des cellules absolues de la suspension cellulaire unique à partir MLN et ensuite préparer les tubes suivantes: le contrôle non marqué (MLN cellules de souris non exposées à l&#39;OVA-FITC), les OVA-FITC de commande, CD11c + de contrôle unique, CD11b + double contrôle, CD11b + OVA-FITC + double contrôle, CD11c + OVA-FITC + double contrôle et CD11c + CD11b échantillons + + OVA-FITC. </li></ol> 5. Les résultats représentatifs Les points dans le temps requis pour la sensibilisation par voie intrapéritonéale à induire une inflammation allergique des voies respiratoires est important et doit être réalisée comme le montre la figure 1. Après une sensibilisation par voie intrapéritonéale et le défi OVA voies respiratoires, afin de confirmer l&#39;induction de l&#39;inflammation des voies allergique, certaines souris peuvent être sacrifiés et les analyses histologiques peuvent être effectués sur les sections du poumon comme le montre la figure 2. Présence de cellules inflammatoires peuvent être confirmés par hématoxyline et éosine (Figure 2A) et la présence de la production de mucus peuvent être évaluées par périodique-acid-Schiff tache (figure 2B). Au total, ce sera de confirmer l&#39;induction de l&#39;inflammation des voies allergique suite à l&#39;OVA défi d&#39;OVA-souris sensibilisées 8. En revanche, les articles du poumon chez les souris traitées au sérum devraient être libres de toute inflammation ainsi que l&#39;absence de toute la production de mucus. En outre, suivant défi OVA, pulmonaire PED UEDN<html> la maturation et la migration RGO après les ganglions lymphatiques drainant. L&#39;analyse des cellules CD11c + dans les ganglions lymphatiques médiastinaux (MLN) à partir de souris sensibilisées et a contesté avec de l&#39;OVA est prévu pour afficher une plus grande proportion de cellules CD11c + par rapport à la solution saline souris traitées (figure 3A). En outre, l&#39;analyse de la numération absolue de CD11c + CD11b + OVA-FITC + cellules dans le MLN de souris OVA-sensibilisés et contestée, devrait montrer une significativement plus élevé (c.-à-plusieurs fois supérieures) compte que les homologues de salins les souris traitées ( La figure 3B). <img alt="Figure 1" src="/files/ftp_upload/4014/4014fig1.jpg" /> Figure 1. Le protocole expérimental pour l&#39;induction de l&#39;ovalbumine (OVA) induit l&#39;inflammation allergique des voies respiratoires chez la souris le long des voies aériennes avec le défi avec FITC étiqueté ovalbumine (OVA-FITC). fig2.jpg &quot;/&gt; Sensibilisation figure 2. OVA suivie par défi voies aériennes OVA conduit à l&#39;induction de voies aériennes inflammation allergique identifiée par coloration d&#39;hématoxyline et éosine de sections du poumon indiqués dans (A) et mène également à la production de mucus dans les voies respiratoires telles qu&#39;identifiées par coloration de Schiff acide périodique des poumons sections comme indiqué dans (B). <img alt="Figure 3" src="/files/ftp_upload/4014/4014fig3.jpg" /> Figure 3. OVA-FITC défi induit la migration DC à partir des poumons vers les ganglions lymphatiques médiastinaux (MLN). (A) des parcelles par cytométrie en flux représentant des proportions de cellules CD11c + dans le MLN de contrôle ou de l&#39;OVA souris sensibilisées. (B) des valeurs absolues de CD11c + + cellules CD11b + OVA-FITC dans le MLN de souris salines ou OVA-sensibilisés. * P &lt;0,05. <a href="http://www.jove.com/files/ftp_upload/4014/4014fig3large.jpg" target="_blank">Cliquez ici pour agrandir la figure</a> .

<ol>
	<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>

Pas de conflits d&#39;intérêt déclarés.

La méthode présentée ici propose une approche basée sur la cytométrie de flux pour analyser les PED pulmonaires, basées sur la livraison d&#39;OVA-FITC pour le défi des voies respiratoires. Ceci permet la surveillance sélective des PED pulmonaires, qui prennent OVA-FITC et par conséquent, les populations de DC, qui sont contrôlés sont effectivement ceux qui participent à la réponse immunitaire des voies respiratoires au cours de l&#39;inflammation induite par OVA allergique des voies respiratoires. Chez les...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Ovalbumin </td><td> Sigma-Aldrich </td><td> A5503 </td></tr><tr><td> Alun </td><td> Thermo Scientific </td><td> 77161 </td></tr><tr><td> OVA-FITC </td><td> Sigma-Aldrich </td><td> A9771 </td></tr><tr><td> Collagénase D </td><td> Roche </td><td> 11088858001 </td></tr><tr><td> Anticorps </td><td> e-Bioscience </td><td></td></tr></tbody></table>

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

Les cellules dendritiques (CD) sont les principaux acteurs impliqués dans l&#39;initiation de la réponse immunitaire adaptative en activant les cellules T spécifiques de l&#39;antigène. PED sont présents dans les tissus périphériques à l&#39;état d&#39;équilibre, mais en réponse à une stimulation antigénique, les CD prennent de l&#39;antigène et rapidement migrer vers les ganglions lymphatiques drainant où ils initient la réponse des cellules T contre l&#39;antigène 1,2. En outre, les PED jouent également un rôle clé dans le déclenchement auto-immune ainsi que la réponse immunitaire allergique 3. PED jouer un rôle essentiel dans l&#39;initiation de la réponse immunitaire et l&#39;induction de la tolérance dans le cadre de 4 environnement du poumon. L&#39;environnement du poumon est largement tolérogène, en raison de l&#39;exposition à la vaste gamme d&#39;antigènes de l&#39;environnement 5. Toutefois, chez certaines personnes il ya une rupture de la tolérance, ce qui conduit à l&#39;induction de l&#39;allergie et l&#39;asthme. Dans cette étude, nous décrivons une stratégie, qui peut être utilisé pour surveiller des voies respiratoires DC maturation et migration en réponse à l&#39;antigène utilisé pour la sensibilisation. La mesure de la maturation des voies respiratoires DC et de la migration permet d&#39;évaluer la cinétique de la réponse immunitaire au cours de l&#39;inflammation allergique des voies respiratoires et aide aussi à comprendre l&#39;ampleur de la réponse immunitaire subséquente ainsi que les mécanismes sous-jacents. Notre stratégie est basée sur l&#39;utilisation de l&#39;ovalbumine comme un agent sensibilisant. Ovalbumin induite par l&#39;asthme allergique est un modèle largement utilisé pour reproduire l&#39;éosinophilie des voies aériennes, l&#39;inflammation pulmonaire et une élévation des taux d&#39;IgE trouvés lors de l&#39;asthme 6,7. Après la sensibilisation, les souris sont contestés par l&#39;administration intranasale d&#39;ovalbumine marquée FITC, ce qui permet d&#39;étiquetage spécifique des PED voies respiratoires ovalbumine absorption qui. Ensuite, en utilisant plusieurs marqueurs spécifiques DC, nous pouvons évaluer la maturation de ces pays en développement et peuvent également évaluer leur migration vers les ganglions lymphatiques drainant en employant la cytométrie en flux.

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

Nous décrivons une stratégie pour surveiller la maturation et la migration des cellules dendritiques pulmonaires en réponse à l&#39;ovalbumine dans le cadre de l&#39;inflammation induite par l&#39;ovalbumine allergique des voies respiratoires. Cette stratégie peut être modifié afin d&#39;évaluer la migration des cellules dendritiques pulmonaires dans les paramètres de l&#39;infection.

L&#39;analyse de la maturation des cellules dendritiques pulmonaire et de la migration au cours de l&#39;inflammation allergique des voies respiratoires

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

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JoVE Journal

Immunology and Infection

14.9K vues.  McMaster University, Hamilton. Nous décrivons une stratégie pour surveiller la maturation et la migration des cellules dendritiques pulmonaires en réponse à l'ovalbumine dans le cadre de l'inflammation induite par l'ovalbumine allergique des voies respiratoires. Cette stratégie peut être modifié afin d'évaluer la migration des cellules dendritiques pulmonaires dans les paramètres de l'infection.

Vidéo: 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.

Human T isolement des lymphocytes, de la Culture et de l&#39;analyse des migrations 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 ...

Recherche

Immunologie et infection

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.

Protocole optimisé pour une transfection efficace des cellules dendritiques, sans maturation cellulaire

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.

Débit analyse par cytométrie de cellules immunitaires dans aortes murin

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.

La migration des cellules dendritiques de surveillance utilisant l&#39;imagerie par résonance 19F / 1H magnétique

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 méthode d&#39;imagerie à distinguer inflammation aiguë et chronique

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.

L&#39;isolement, la caractérisation et de l&#39;examen fonctionnel du Réseau de cellules gingivale immunitaire

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.

Administration intra-trachéale de<em&gt; Haemophilus influenzae</em&gt; Dans les modèles de souris pour étudier Airway Inflammation

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 test basé CFSE pour étudier la migration de la peau murine cellules dendritiques dans les ganglions lymphatiques de drainage lors de l&#39;infection avec<em&gt; Mycobacterium bovis</em&gt; Bacille 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.

coupe de précision de la souris Tranches Lung pour visualiser les cellules vivantes pulmonaire dendritiques

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.

Analyse de cisaillement induite par le flux de Migration des cellules B Zone marginale murin 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, ...