Nous tenons à remercier M. Höxter d&#39;assistance technique dans le tri primaire AECII murin de niveau de biosécurité 2 échantillons. Ce travail a été soutenu par des subventions de la Fondation allemande de recherche (DFG) à DB (SFB587, TP B12 et BR2221/1-1) et une bourse de l&#39;École Hanovre recherche biomédicale (DFG CGC 108) à AA. DB est soutenu par l&#39;Initiative du Président et du Fonds de réseautage de l&#39;Association Helmholtz des centres de recherche allemands (HGF), sous le numéro de contrat W2/W3-029.

Les détails concernant les réactifs et les matériaux requis sont répertoriés dans le tableau à la fin du protocole ci-dessous. Avant de commencer à travailler, préparer des tubes de 15 ml (un par souris) contenant 4 ml de dispase et préchauffer les à 37 ° C dans un bain-marie. Dans un bloc de chauffage, chauffer rapidement de petites aliquotes de 1% d&#39;agarose bas point de fusion (dans l&#39;eau) à 95 ° C jusqu&#39;à ce que liquéfié et ensuite refroidir à 45 ° C jusqu&#39;à utilisation. 1. Préparation du poumon de souris <ol><li> Sacrifiez la souris par le CO 2 asphyxie. Remarque: Ne pas effectuer dislocation cervicale car cela blesser la trachée afin que les étapes suivantes du protocole, tels que l&#39;installation du poumon avec le liquide, ne peut pas être réalisée avec succès. Veiller à une perte des réflexes nociceptifs. </li><li> Pulvériser la souris avec de l&#39;éthanol et de faire une longue incision le long de la ligne médiane ventrale du corps. Tirez le pelage ventral et la peau et par la suite aussi bien couper et enlever le péritoine. </li><li> Exsangue l&#39;souris en coupant la veine jugulaire droite et gauche (veine jugulaire), ainsi que l&#39;artère rénale (renalis Arteria). Retirez le sang qui coule avec le tissu. </li><li> Attentivement la perforation, puis retirer la membrane pour exposer le coeur et les poumons en coupant les côtes. Faites attention de ne pas blesser le poumon. </li><li> Avec une canule 26G et une seringue de 10 ml remplie de phosphate froid (PBS), la perforation du ventricule droit du cœur et le poumon perfuser avec du PBS jusqu&#39;à disparition de sang. </li><li> Couper et enlever les glandes salivaires pour exposer la trachée. Aussi soigneusement coupé les muscles entourant la trachée. </li><li> Insérez une canule à demeure 22G dans la trachée, retirez l&#39;aiguille et pousser le cathéter en plastique vers le poumon. Fixer le cathéter en attachant un petit morceau de fil autour de la trachée et du cathéter. </li></ol> 2. La digestion enzymatique du tissu pulmonaire Si vous le souhaitez, un échantillon de fluide de lavage bronchoalvéolaire peut êtrepréparé par rinçage les poumons par l&#39;intermédiaire du cathéter inséré dans la trachée ou avec du PBS support avant l&#39;étape suivante. <ol><li> En utilisant une seringue de 2 ml, soigneusement insuffler un volume maximal de 2 ml de dispase (à partir de l&#39;aliquote de 4 ml) dans les poumons à travers le cathéter de sorte que tous les lobes sont pleinement développées. Échanger la seringue avec une seringue de 1 ml contenant 0,5 ml de la gélose liquéfiée et aussi instiller dans les poumons. </li><li> Laissez la seringue sur le cathéter pour empêcher le reflux de l&#39;agarose et couvrir immédiatement les poumons avec du papier absorbant de laboratoire et de la glace. Laissez le gel d&#39;agarose pour un couple de minutes. </li><li> Retirer le papier de soie, de la glace, la seringue et le cathéter, couper la trachée et de l&#39;accise du poumon, le cœur et le thymus de la poitrine. Ensuite, rincez le poumon dans un plat avec du PBS et retirer le cœur, le thymus et la trachée reste. </li><li> Mettez le poumon dans le reste de 2 ml de dispase et incuber à température ambiante pendant 45 minutes. </li></ol> 3. Préparation du poumonSuspension cellulaire <ol><li> Retirez le poumon de la dispase et le transférer dans un plat contenant 7 ml de anti-CD16/32 anticorps (1 pg / ml de concentration finale) dans du milieu DMEM et 100 ul de DNase. </li><li> En utilisant des pinces, complètement désintégrer le tissu pulmonaire en le tirant à l&#39;écart et laisser incuber pendant 10 min à température ambiante tout en secouant délicatement sur une bascule réglé à 200 tours par minute. Si vous le souhaitez, la suspension de cellules peuvent ensuite être stockés à 4 ° C jusqu&#39;à l&#39;étape suivante. </li><li> Filtrer la suspension de cellules à travers les mailles de nylon abord avec 100 um et par la suite avec 70 um, 48 um et 30 um pores. Prendre soin de laver chaque maille ainsi que les tubes plats et à fond avec du DMEM pour minimiser la perte de cellules et à maximiser le rendement. Si vous êtes mise en commun des échantillons, cela peut être réalisé ici par la suite, passant suspensions de cellules de souris d&#39;un groupe à travers le même filtre. Remplacement du filtre en cas de colmatage. </li><li> En fonction du volume, de transférer le filtrat à un ou plusieurs tubes de 50 ml etcentrifuger pendant 15 min à 160 xg et 4 ° C. Enlever le surnageant. </li><li> Remettre en suspension le culot cellulaire dans 2 ml de tampon de lyse des érythrocytes (0,15 M NH 4 Cl, 0,01 M KHCO 3, 0,1 mM EDTA, pH 7,2) et mettre fin rapidement lyse par addition de 13 ml de milieu DMEM. Transférer la solution dans un tube de 15 ml et centrifuger pendant 12 min à 160 xg et 4 ° C. </li></ol> 4. La coloration d&#39;anticorps de flux cellulaire par cytométrie de tri <ol><li> Reprendre les cellules dans 3 ml de cocktail d&#39;anticorps primaire, préparée dans du milieu DMEM à des dilutions appropriées pour la cytométrie en flux. (Anticorps ont été préalablement titrée pour les dilutions de travail optimales par coloration 1 x 10 6 splénocytes dans un volume de 100 pl. Utiliser 3 ml de cocktail d&#39;anticorps primaire contenant des concentrations optimales pour chacun des anticorps utilisés pour les cellules mises en commun d&#39;un maximum de cinq souris. Si plus de souris sont mises en commun pour un échantillon, régler le volume). </li><li> Pour la coloration, incuber les cellules pendant 10 min dans l&#39;obscuritéà 4 ° C. </li><li> Laver les cellules en remplissant le tube de 15 ml avec du milieu DMEM et centrifugation pendant 12 min à 160 xg et 4 ° C. </li><li> Dans le cas des anticorps biotinylé ou découplées ont été utilisés dans 4.1: Enlever le surnageant et remettre en suspension les cellules dans 2 à 3 ml cocktail d&#39;anticorps secondaire. Colorer pendant 10 min à 4 ° C dans l&#39;obscurité. </li><li> Laver les cellules à travers le remplissage du tube avec du DMEM et centrifugation pendant 12 min à 160 xg et 4 ° C. </li><li> Remettre en suspension les cellules dans 1 ml de DMEM et de pré-filtre à travers un filtre 50 um dans un tube de tri cellulaire. En option: Compter les cellules pour obtenir le nombre total de cellules dans la suspension de cellules du poumon. </li></ol> 5. Tri cellulaire <ol><li> Mélanger les cellules au vortex avant triage. Utiliser une buse de 100 um de tri cellulaire de AECII. Pression de fluide de gaine ainsi que des longueurs d&#39;onde d&#39;émission du laser et de détection dépend de l&#39;instrument utilisé pour l&#39;écoulement de tri cellulaire par cytométrie de même que les fluorochromes utilisés dans le ANTIBody procédure de coloration. </li><li> Porte sur les cellules CSE élevés qui sont négatifs pour les fluorochromes utilisés pour la coloration et le tri dans un tube contenant du DMEM. Utiliser avant la hauteur de dispersion (FSC-H) vs zone (FSC-A) et la hauteur de dispersion latérale (SSC-H) vs zone (SSC-A) fenêtres afin d&#39;exclure tout doublets ou des agrégats de cellules (voir les détails de la stratégie de porte à Figure 1 a). </li><li> Afin de recueillir la centrifugeuse triés cellules pendant 20 min à 280 xg et 4 ° C. </li><li> Remettre en suspension les cellules dans la culture ou de support d&#39;un tampon tel que requis pour une analyse ultérieure. </li></ol>

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Pharmacol. 82, 548-555 (2011).</li><li>Zarbock, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+surfactant+protein+C+mutation+A116D+alters+cellular+processing,+stress+tolerance,+surfactant+lipid+composition,+and+immune+cell+activation.">The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation.</a> BMC. Pulm. Med. 12, 15 (2012).</li><li>Taubenberger, J. K., Morens, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathology+of+influenza+virus+infections.">The pathology of influenza virus infections.</a> Annu. Rev. Pathol. 3, 499-522 (2008).</li><li>Dobbs, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+alveolar+type+II+cells.">Isolation and culture of alveolar type II cells.</a> Am. J. Physiol. 258, L134-L147 (1990).</li><li>Corti, M., Brody, A. R., Harrison, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+primary+culture+of+murine+alveolar+type+II+cells.">Isolation and primary culture of murine alveolar type II cells.</a> Am. J. Respir. Cell Mol. Biol. 14, 309-315 (1996).</li><li>Beers, M. F., Kim, C. Y., Dodia, C., Fisher, A. 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Lung Res. 17, 985-995 (1991).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiated+human+alveolar+type+II+cells+secrete+antiviral+IL-29+(IFN-lambda+1)+in+response+to+influenza+A+infection.">Differentiated human alveolar type II cells secrete antiviral IL-29 (IFN-lambda 1) in response to influenza A infection.</a> J. Immunol. 182, 1296-1304 (2009).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+response+to+influenza+A+virus+in+differentiated+human+alveolar+type+II+cells.">Innate immune response to influenza A virus in differentiated human alveolar type II cells.</a> Am. J. Respir. Cell Mol. Biol. 45, 582-591 (2011).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Notre protocole pour l&#39;isolement des murin AECII par cytométrie en flux offre un moyen rapide d&#39;accéder à des cellules primaires du poumon de souris pour toute une série d&#39;études fonctionnelles et moléculaires. La procédure décrite donne populations très viables et pure de AECII qui sont en nombre suffisant pour les analyses ultérieures directes, telles que l&#39;ARN isolés (voir figure 2b) et l&#39;étude du transcriptome. Pour les applications fonctionnelles, il est également p...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> demeure canule 22G Introcan </td><td> Braun </td><td> REF 4252098B </td><td></td></tr><tr><td> Dispase, 100 ml (5000 unités caséinolytiques) </td><td> BD Biosciences </td><td> 354235 </td><td> aliquote de 4 ml en tubes de 15 ml, à -20 ° C </td></tr><tr><td> Plaque Biozym Agarose </td><td> Biozym </td><td> 840101 </td><td> 1% p / v dans H 2 O </td></tr><tr><td> Désoxyribonucléase I de pancréas bovin, 2000 unités Kunitz / flacon </td><td> Sigma-Aldrich </td><td> D4263 </td><td> fraîchement dissoudre le contenu de 1 flacon dans 300 ul de DMEM </td></tr><tr><td> DMEM </td><td> Gibco </td><td> 22320-022 </td><td> utilisé tel que prévu par le fabricant (Low Glucose, pyruvate, HEPES) </td></tr><tr><td> crépines cellulaires (100 um, 75 um) </td><td> BD Falcon </td><td> 352360, 352350 </td><td></td></tr><tr><td> filet de nylon (48 um, 30 um) </td><td> Buckmann GmbH </td><td> 03-48/26-1020, 03-30/18-108 </td><td></td></tr><tr><td> CellTrics filtre 50 um </td><td> PARTEC </td><td> 04-0042-2317 </td><td></td></tr><tr><td> anti-souris CD16/CD32 </td><td> BioLegend </td><td> 101302 </td><td> clone 93; purifié </td></tr><tr><td> anti-souris F4/80 </td><td> BioLegend </td><td> 123116 </td><td> clone BM8; APC couplage </td></tr><tr><td> anti-souris CD11b </td><td> BioLegend </td><td> 101208 </td><td> clone M1/70; PE couplée </td></tr><tr><td> anti-souris CD11c </td><td> BioLegend </td><td> 117310 </td><td> clone N418, couplé APC </td></tr><tr><td> anti-sourisCD45 </td><td> BioLegend </td><td> 103102 </td><td> clone 30-F11; purifié </td></tr><tr><td> anti-CD19 de souris </td><td> eBioscience </td><td> 12-0193-83 </td><td> eBio 1D3; PE couplage </td></tr><tr><td> polyclonal de chèvre anti-IgG de rat </td><td> BD Pharmingen </td><td> 550767 </td><td> polyclonal, PE couplage </td></tr><tr><td></td><td></td><td></td><td> Anticorps couplés à des fluorochromes alternatives peuvent être utilisées, selon le cytomètre de flux et de lasers disponibles. </td></tr></tbody></table>

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

Au cours des dernières années, la contribution des cellules alvéolaires de type II épithéliales (AECII) à divers aspects de la régulation du système immunitaire dans le poumon a été de plus en plus reconnue. AECII Il a été démontré à participer à la production de cytokines dans les voies respiratoires enflammées et même agir comme cellules présentatrices d&#39;antigène à la fois l&#39;infection et des lymphocytes T auto-immunité médiée par 1-8. Par conséquent, ils sont particulièrement intéressants dans les contextes cliniques tels que l&#39;hyper-réactivité des voies aériennes à l&#39;étranger et des auto-antigènes ainsi que les infections qui ciblent directement ou indirectement AECII. Cependant, notre compréhension des fonctions immunologiques détaillées desservis par les cellules alvéolaires de type II épithéliales des poumons en bonne santé ainsi que dans l&#39;inflammation reste fragmentaire. De nombreuses études concernant la fonction AECII sont effectuées à l&#39;aide de la souris ou l&#39;homme lignées de cellules épithéliales alvéolaires 9-12. Travailler avec des lignées de cellules offre certes de nombreux avantages, tels que la disponibilité d&#39;un grand nombre of cellules pour des analyses approfondies. Cependant, nous croyons que l&#39;utilisation de AECII murin primaire permet une meilleure compréhension du rôle de ce type de cellules dans des processus complexes comme l&#39;infection ou une inflammation auto-immune. Primaire AECII murin peut être isolé directement à partir d&#39;animaux qui souffrent de ces affections respiratoires, ce qui signifie qu&#39;ils ont été soumis à tous les autres facteurs extrinsèques jouent un rôle dans la mise en analysés. A titre d&#39;exemple, AECII viable peut être isolé à partir des souris infectées par voie intranasale avec le virus de la grippe A, qui touche principalement les cellules à la réplication 13. En outre, grâce infection ex vivo de AECII isolés de souris saines, l&#39;étude des réponses cellulaires montés lors de l&#39;infection peut être prolongée. Notre protocole pour l&#39;isolement de AECII murin primaire est basée sur la digestion enzymatique de la souris suivies par les poumons d&#39;étiquetage de la suspension cellulaire obtenue avec des anticorps spécifiques de CD11c, CD11b, F4/80,CD19, CD45 et CD16/CD32. AECII granulaires sont ensuite identifiés comme non marqué et la dispersion latérale élevée (SSC haut) et population de cellules sont séparées par tri cellulaire activé par fluorescence 3. En comparaison à d&#39;autres méthodes d&#39;isolement de cellules primaires épithéliales de poumons de souris, notre protocole d&#39;isolement par cytométrie de flux AECII par sélection négative donne intacte, très viable et pur AECII dans un temps relativement court. En outre, et contrairement aux méthodes conventionnelles d&#39;isolement par le panoramique et l&#39;épuisement des lymphocytes par l&#39;intermédiaire de la liaison de l&#39;anticorps couplés billes magnétiques 14, 15, cytométrie en flux de tri cellulaire permet la discrimination par le biais de la taille des cellules et la granularité. Étant donné que l&#39;instrumentation pour le tri cellulaire par cytométrie en flux est disponible, la procédure décrite peut être appliquée à des coûts relativement faibles. Suivant les anticorps et les enzymes standards pour la désintégration du poumon, pas de réactifs supplémentaires tels que magnétiqueperles sont nécessaires. Les cellules isolées sont adaptés à un large éventail d&#39;études fonctionnelles et moléculaires, notamment la culture in vitro et des essais de stimulation des cellules T ainsi que transcriptome, du protéome ou des analyses sécrétome 3, 4.

Lors du tri des suspensions de cellules pulmonaires isolées à partir de souris saines, la porte sera AECII représentent généralement environ 42 ± 10% de tous les événements. Ce pourcentage peut être nettement plus faible lorsque les souris atteintes de maladies respiratoires telles que une infection virale sont utilisées, comme la suspension cellulaire initiale contient une proportion considérablement plus élevée de lymphocytes et d&#39;autres cellules immunitaires recrutées pour les voies respiratoires. P...

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

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

Nous décrivons l&#39;isolement rapide des primaires murins cellules alvéolaires de type II épithéliales (AECII) par flux cytométrique sélection négative. Ceux-ci montrent AECII viabilité et à la pureté et conviennent à un large éventail d&#39;études fonctionnelles et moléculaires concernant leur rôle dans les affections respiratoires comme les maladies auto-immunes ou infectieuses.

Isolation par cytométrie en flux des cellules primaires murins épithéliales alvéolaires de type II pour les études fonctionnelles et moléculaires

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

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Immunology and Infection

26.1K vues.  Helmholtz Centre for Infection Research. Nous décrivons l'isolement rapide des primaires murins cellules alvéolaires de type II épithéliales (AECII) par flux cytométrique sélection négative. Ceux-ci montrent AECII viabilité et à la pureté et conviennent à un large éventail d'études fonctionnelles et moléculaires concernant leur rôle dans les affections respiratoires comme les maladies auto-immunes ou infectieuses.

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

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

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

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

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 ...

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Immunologie et infection

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

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

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

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

Analyse de l&#39;altération de l&#39;épithélium Produit par<em&gt; Entamoeba histolytica</em&gt; Maladies infectieuses

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

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

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

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

Isolement, identification et la purification de cellules murines thymique épithéliales

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

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

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

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

Isolement et injection intraveineuse de moelle osseuse de souris dérivées monocytes

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

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

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

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

Isolement et analyse par cytométrie en flux des cellules immunitaires du cerveau ischémique souris

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

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

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

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

L&#39;isolement et la cytométrie en flux analyse des cellules mononucléaires du sang périphérique gliome infiltrant

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

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

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

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

L&#39;isolement et la caractérisation par cytométrie en flux de l&#39;intestin grêle Lymphocytes murine

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

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

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

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

Isolement et cytométrie de flux Analyse de Human Endocervical Gamma Delta cellules T

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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

Isolement et la Culture In Vitro de Macrophages alvéolaires murines et humaines

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

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

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

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

Digestion du foie murin pour une cytométrie de cellules endothéliales lymphatiques

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