Nous tenons à remercier Aaron Rae (Emory University Département de pédiatrie et des soins de santé pour enfants de Core flux Atlanta) pour le tri cellulaire. Ce travail a été soutenu par le NIH subvention AA01787001, une bourse de perfectionnement de carrière de la maladie de Crohn et la colite Foundation of America, et un enfants Emory-Egleston de recherche de semences Centre subvention pour TLD

1. Dissection et dissociation des cellules épithéliales intestinales Préparation des réactifs et du matériel: <ul><li> Chaude Ca 2 + / Mg 2 + sans PBS (FMC PBS) à la température ambiante. </li><li> Chaud Ca 2 + / Mg 2 +-libres HBSS avec 5% de FBS (HBSS CMF / FBS) et 2 mM EDTA à température ambiante. </li><li> Chaud Agitateur orbital à 37 ° C. </li></ul> Remarque: Les étapes 1.1 à 1.7 doit être effectuée aussi rapidement que possible afin de minimiser l&#39;étendue de la mort cellulaire et pour obtenir un rendement cellulaire maximale. <ol><li> Euthanasier les souris dans un chambre de CO 2 et d&#39;éthanol de pulvérisation de 70% sur l&#39;abdomen et du thorax. </li><li> Faire une petite incision horizontale au milieu de l&#39;abdomen avec une paire de ciseaux et peler la peau pour exposer le péritoine. </li><li> Passez à séparer l&#39;estomac de l&#39;intestin grêle supérieur en coupant au niveau du sphincter pylorique. Tease loin du mésentère avec des pinces et couper de nouveau àde la valvule iléo-caecale pour libérer tout l&#39;intestin grêle du gros intestin. Faites une entaille à la marge de l&#39;anus et encore démêler le mésentère jusqu&#39;à ce que le gros intestin est libre. </li><li> Couper le côlon longitudinalement l&#39;aide de ciseaux et rincer le contenu fécaux et de mucus dans la lumière intestinale dans le CMF PBS à température ambiante. </li><li> Utilisez des ciseaux et des pinces à disséquer macroscopiquement les plaques de Peyer le long de la surface anti-mésentérique de l&#39;intestin grêle et coupés de l&#39;intestin grêle longitudinalement. </li><li> Laver les lumière de l&#39;intestin grêle des matières fécales et de mucus dans CMF PBS à température ambiante. </li><li> Par ailleurs coupé l&#39;intestin grêle / de grande taille en pièces d&#39;environ 1,5 cm et les placer dans des tubes séparés 50 ml coniques contenant 30 ml de pré-chauffée HBSS CMF / FBS et 2 mM EDTA. Ne pas ajouter plus de 1 intestin par 50 ml tube conique. </li><li> Horizontalement placer chaque tube conique de 50 ml dans un agitateur orbital et agiter pendant à 250 rpm pendant 20 min à 37 ° C &lt;/ Li&gt; <li> Placez une passoire unique fil sur un seau de déchets et verser le contenu de chaque tube conique de 50 ml par de récupérer les 1,5 pièces cm de l&#39;intestin et les placer dans différents tubes coniques de 50 ml contenant 30 ml de préchauffées CHBSS / FB avec 2 mM d&#39;EDTA. </li><li> Répétez 1.8. </li></ol> 2. Digestion du tissu et l&#39;isolement des cellules intestinales Préparations à base de réactifs et d&#39;équipements: <ul><li> Solution de collagénase: 1,5 mg / ml Type VIII collagénase dissous dans préchauffé HBSS CMF / FBS avec 40 pg / ml de DNase I. </li><li> Préchauffé HBSS CMF / FBS et 2 mM EDTA. </li><li> Préchauffé agitateur orbital à 37 ° C. </li><li> Ice-froid HBSS CMF / FBS. </li></ul><ol><li> Après le second tour de la secousse, versez les contenus de chaque tube de 50 ml conique à travers le filtre et transférer 1,5 pièces cm de l&#39;intestin à un petit plastique pèsent bateau après tamponnant loin des médias en excès en utilisant une serviette en papier. </li><li> Rapidement émincer le 1.5 Pièces cm de l&#39;intestin à l&#39;aide de ciseaux directement dans le bateau de poids et d&#39;ajouter l&#39;intestin hachées 20 ml de solution de collagénase. Horizontalement placer chaque tube conique de 50 ml dans un agitateur orbital et faire digérer à 200 rpm pendant 10-20 min à 37 ° C. S&#39;il vous plaît voir ci-dessous portant sur ​​l&#39;optimisation. </li><li> En bref vortex pour s&#39;assurer la dissociation complète de tout tissu restant intestinale et le filtre à travers une passoire 100 um cellules directement dans un tube de 50 ml conique. </li><li> Haut de chaque tube conique de 50 ml avec du HBSS CMF / FBS et centrifuger à 1500 rpm pendant 5 min à 4 ° C. Si un solide culot n&#39;est pas observée pour les échantillons du côlon après centrifugation, les échantillons doivent être centrifugés à nouveau de 3,5 min. Répétez cette étape de lavage une fois de plus. </li><li> Décanter le surnageant et remettre en suspension le culot cellulaire dans glacée HBSS CMF / FBS et des échantillons de placer sur la glace. </li><li> Passez à la section 3 pour l&#39;acquisition de FACS / analyse ou de la section 4 pour magnétique talon d&#39;enrichissement pour la cellule haute-vitesse Sorting. </li></ol> 3. Coloration des anticorps pour l&#39;analyse des flux multi-couleur par cytométrie de pays en développement et les macrophages Préparations à base de réactifs et d&#39;équipements: <ul><li> Ice-froid CMF PBS. </li><li> Tampon de coloration glacée (CMF PBS + 5% de FBS). </li><li> Préparer cellules mortes tache dans glacée CMF PBS à 1:1000 dilution à l&#39;aide LIVE / DEAD Kit fixable Aqua Dead Cell taches. </li><li> Préparer le cocktail d&#39;anticorps coloration en ajoutant les suivantes marquées par fluorescence des anticorps monoclonaux (mAb) à la glace froide tampon de coloration: CD45-PerCP, CD103-PE, CD11c-APC, CMH-II (IA b)-Alexa Fluor 700, CD11b -eFluor 450, F4/80-PE-Cy7. </li></ul><ol><li> Transfert des cellules dans un 5 ml polystyrène à fond rond (FACS) du tube. </li><li> Laver les cellules deux fois dans la glace froide CMF PBS. </li><li> Échantillons Incuber avec cellule morte tache pendant 15 min sur la glace dans l&#39;obscurité. </li><li> Laver les cellules deux fois dans la glace froide CMF PBS. </li><li> Cellules de bloc avec 2.4G2 anti-FcγRIII/II dans glacée colorationtampon pendant 10 min sur la glace. </li><li> Laver les cellules dans un tampon de coloration glacée. </li><li> Incuber les échantillons avec un cocktail d&#39;anticorps coloration pendant 20 min sur la glace dans l&#39;obscurité. </li><li> Laver les cellules avec du tampon glacé coloration à deux reprises et remettre en suspension des échantillons dans 400 ul de tampon glacé coloration et passer à travers bouchon 40 um filtre sur tubes FACS. </li><li> Acquérir des échantillons sur LSR II cytomètre (BD) tels que définis par la stratégie de porte à la section 5 et la figure 1. </li></ol> 4. Enrichissement des pays en développement et les macrophages de l&#39;intestin Préparations à base de réactifs et d&#39;équipements: <ul><li> Tampon de coloration glacée (CMF PBS + 5% de FBS). </li></ul><ol><li> Incuber la suspension cellulaire unique obtenue à l&#39;étape 2.5 avec CD11b et CD11c perles MACS selon les instructions du fabricant. </li><li> Laver les cellules avec un tampon de coloration glacée suivie d&#39;une centrifugation. </li><li> Rejeter le surnageant et remettre en suspension le culot cellulaire dans 1 ml de tampon coloration glacéeet passer à travers une passoire 100 um cellulaire suivie d&#39;une passoire cellule 40 um. </li><li> Enrichir pour billes magnétiques joints par sélection positive des cellules en utilisant MACS LS colonne magnétique. </li><li> Répétez l&#39;étape 4.2 et éliminer le surnageant. </li><li> Incuber les cellules avec des mAbs marqueurs de surface tels que décrits à l&#39;étape 3.7. </li><li> Lavez-bille magnétique enrichis cellules deux fois avec tampon de coloration glacée. Remettre en suspension les culots de cellules dans 500 ul tampon de coloration glacée sans l&#39;azoture de sodium, et de passer à 40 um tamis cellulaire dans un tube FACS. </li><li> Passez à la FACS-tri sur la trieuse de BD ARIA II cellulaire pour trier DC intestinale et / ou sous-ensembles de macrophages d&#39;intérêt. </li></ol> 5. Stratégie d&#39;ouverture de porte pour LP APC Note: S&#39;il vous plaît noter que les cellules intestinales non colorées peuvent être utilisées comme contrôle négatif pour aider à la mise en place correcte des portes pour séparer les populations positives et négatives. <ol><li> Comme le montre la figure1, créez un tracé de points et exclure les cellules qui sont positives pour la cellule morte tache (fig. 1A), suivie par l&#39;exclusion d&#39;événements doublet (Fig. 1B et C). Puis, la porte sur les cellules d&#39;intérêt en fonction de transmettre et de diffusion latérale en veillant à exclure les débris (Fig. 1D). </li><li> Créez un autre tracé de points et porte en outre sur les CD45 + et IA b + cellules, ce qui caractérise phénotypiquement APC (Fig. 1E). </li><li> Sur un tracé de points distincts, d&#39;analyser et d&#39;expression pour CD11b CD11c de distinguer DC spécifique et sous-ensembles des macrophages (R1, R2 et R3; Fig. 1F). Les cellules de R1 sont CD11c + CD11b ternes / - cellules. La région, R2, est délimité de telle sorte que ces cellules ont des niveaux similaires de l&#39;expression en surface de CD11c comme dans les cellules de R1, mais les cellules de R2 expriment également CD11b. Par la suite, la région R3 est désigné pour les cellules qui sont + CD11b et terne / CD11c -. </li><li> Unalyze régions R1, R2, R3 et autre expression pour F4/80 et CD103 pour différencier des macrophages et des populations de DC, respectivement. Le CD11c + CD11b terne / - cellules expriment des niveaux de R1 élevés de l&#39;intégrine αE, CD103, et de faibles niveaux de F4/80 (Fig. 1G). CD11b + CD11c + cellules de R2 sont composés de deux contrôleurs et des macrophages sur la base de leur expression dichotomique de CD103 et F4/80 (figure 1H). Enfin, CD11b + CD11c ternes / - cellules de macrophages constituent R3 basées sur le profil phénotypique de F4/80 + et CD103 - (Fig. 1I) 16. </li></ol> 6. Les résultats représentatifs <img alt="Figure 1" src="/files/ftp_upload/4040/4040fig1.jpg" /> Figure 1. Stratégie d&#39;ouverture de porte pour les PED intestinales et les macrophages. Cellules mortes (A) et doublets (B et C) ont d&#39;abord été exclus de l&#39;analyse, puis petite intestinalecellules internes ont fermée en conséquence de transmettre et de côté de dispersion (D), et CPA ont été défini comme CD45 + b + IA (E). Les macrophages et les PED ont été identifiés par l&#39;expression de CD11b et CD11c (F). CD103 et F4/80 expression pour les cellules pré-dépendants sur R1 (G), R2 (H) et R3 (I), les populations ont été analysées. <img alt="Figure 2" src="/files/ftp_upload/4040/4040fig2.jpg" /> Figure 2. Rendement cellulaire et qualité de la coloration d&#39;anticorps dépend du temps de digestion. CD11b et CD11c motif de coloration et le rendement total de cellules de la sous-(A, D), de façon optimale (B, E) ou sur-digéré (C, F) du tissu intestinal. Les cellules intestinales ont été isolés à partir d&#39;un intestin souris C57BL / 6 petites et pays en développement et les macrophages ont été analysées par FACS sur la BD LSR II. Tension et de compensation ont été définies à l&#39;aide non colorées et seul fluorochrome tachés de splénocytes. Les cellules mortes (Fig. 1A) et doublets (Fig. 1B et C) ont d&#39;abord été exclus del&#39;analyse. Les cellules d&#39;intérêt ont ensuite été analysées en fonction de transmettre et le côté de dispersion (Fig. 1D), suivie par de déclenchement sur ​​CD45 + et IA b + cellules (Fig. 1E). Par la suite, CD11b et CD11c expression a été évaluée chez le CD45 + b + IA cellules pour délimiter trois régions (R1, R2 et R3; Fig. 1F). CD103 et F4/80 expression dans les trois régions a été évaluée à la distinction entre les pays en développement et les macrophages, respectivement. Le CD11c + CD11b / terne - les cellules de R1 ont exprimé des niveaux élevés de l&#39;intégrine αE, CD103, et de faibles niveaux de F4/80 (Fig. 1G). CD11b + CD11c + cellules de R2 ont été composé de deux contrôleurs de domaine et les macrophages en fonction de leur expression et dichotomique de CD103 F4/80 (Fig. 1H), tandis que CD11b + CD11c ternes / - cellules de macrophages constituent R3 basées sur le profil phénotypique de F4 / 80 + et CD103 - ( <strong&gt; Fig. 1I) 16. Les macrophages au sein de la grille R2 et macrophages dans la porte avant R3 ont similaire et propriétés de diffusion secondaires et se distinguent par l&#39;expression CD11c. La dichotomie fonctionnelle de ces sous-ensembles reste incomplètement comprise. La relation entre la durée de la digestion du tissu sur le rendement total de cellules et l&#39;expression de CD11b et CD11c est illustré à la figure 2. Tissu intestinal qui a été digéré pendant 3 min (sous-digestion) a donné le faible nombre total de cellules (Fig. 2D) et les PED et les macrophages donc peu disponibles pour la caractérisation (figure 2A). Digestion du tissu pour 11 min a produit un rendement robuste de cellules vivantes (Fig. 2E) avec les populations de pays en développement et les macrophages qui expriment des niveaux élevés de CD11b et CD11c et phénotypiquement distincte (Fig. 2C). En revanche, la digestion pendant 50 min (plus-digestion) a entraîné un rendement cellule similaire en cas de compared à la digestion optimisée (Fig. 2E et F), cependant, la délimitation des différentes populations de cellules en utilisant CD11b et CD11c est devenu plus obscur que l&#39;expression de CD11c diminuée (Fig. 2C) et le nombre de cellules mortes a augmenté (données non présentées).

<ol>
	<li>Maloy, K. J., Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+homeostasis+and+its+breakdown+in+inflammatory+bowel+disease.">Intestinal homeostasis and its breakdown in inflammatory bowel disease.</a> Nature. 474, 298-306 (2011).</li><li>Nagler-Anderson, C., Terhoust, C., Bhan, A. K., Podolsky, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+antigen+presentation+and+the+control+of+tolerance+and+immunity.">Mucosal antigen presentation and the control of tolerance and immunity.</a> Trends Immunol. 22, 120-122 (2001).</li><li>Abraham, C., Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+the+host+innate+immune+system+and+microbes+in+inflammatory+bowel+disease.">Interactions between the host innate immune system and microbes in inflammatory bowel disease.</a> Gastroenterology. 140, 1729-1737 (2011).</li><li>Macdonald, T. T., Monteleone, I., Fantini, M. C., Monteleone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regula+tine.">Regula tine.</a> Gastroenterology. 140, 1768-1775 (2011).</li><li>Rescigno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+dendritic+cells.">Intestinal dendritic cells.</a> Adv. Immunol. 107, 109-138 (2010).</li><li>Platt, A. M., Bain, C. C., Bordon, Y., Sester, D. P., Mowat, 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=An+independent+subset+of+TLR+expressing+CCR2-dependent+macrophages+promotes+colonic+inflammation.">An independent subset of TLR expressing CCR2-dependent macrophages promotes colonic inflammation.</a> J. Immunol. 184, 6843-6854 (2010).</li><li>Coombes, J. L., Powrie, F. <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+in+intestinal+immune+regulation.">Dendritic cells in intestinal immune regulation.</a> Nat. Rev. Immunol. 8, 435-446 (2008).</li><li>Kelsall, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+understanding+the+phenotype+and+function+of+intestinal+dendritic+cells+and+macrophages.">Recent progress in understanding the phenotype and function of intestinal dendritic cells and macrophages.</a> Mucosal Immunol. 1, 460-469 (2008).</li><li>Pulendran, B., Tang, H., Denning, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Division+of+labor,+plasticity,+and+crosstalk+between+dendritic+cell+subsets.">Division of labor, plasticity, and crosstalk between dendritic cell subsets.</a> Curr. Opin. Immunol. 20, 61-67 (2008).</li><li>Denning, T. L., Wang, Y. C., Patel, S. R., Williams, I. R., Pulendran, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lamina+propria+macrophages+and+dendritic+cells+differentially+induce+regulatory+and+interleukin+17-producing+T+cell+responses.">Lamina propria macrophages and dendritic cells differentially induce regulatory and interleukin 17-producing T cell responses.</a> Nat. Immunol. 8, 1086-1094 (2007).</li><li>Niess, 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=CX3CR1-mediated+dendritic+cell+access+to+the+intestinal+lumen+and+bacterial+clearance.">CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance.</a> Science. 307, 254-258 (2005).</li><li>Milling, S. W., Cousins, L., MacPherson, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+DCs+interact+with+intestinal+antigens.">How do DCs interact with intestinal antigens.</a> Trends Immunol. 26, 349-352 (2005).</li><li>Bilsborough, J., Viney, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+dendritic+cells+play+a+role+in+immunity,+tolerance,+and+disease.">Gastrointestinal dendritic cells play a role in immunity, tolerance, and disease.</a> Gastroenterology. 127, 300-309 (2004).</li><li>Stagg, A. J., Hart, A. L., Knight, S. C., Kamm, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dendritic+cell:+its+role+in+intestinal+inflammation+and+relationship+with+gut+bacteria.">The dendritic cell: its role in intestinal inflammation and relationship with gut bacteria.</a> Gut. 52, 1522-1529 (2003).</li><li>Medina-Contreras, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CX3CR1+regulates+intestinal+macrophage+homeostasis,+bacterial+translocation,+and+colitogenic+Th17+responses+in+mice.">CX3CR1 regulates intestinal macrophage homeostasis, bacterial translocation, and colitogenic Th17 responses in mice.</a> J. Clin. Invest. 121, 4787-4795 (2011).</li><li>Denning, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Specializations+of+Intestinal+Dendritic+Cell+and+Macrophage+Subsets+That+Control+Th17+and+Regulatory+T+Cell+Responses+Are+Dependent+on+the+T+Cell/APC+Ratio,+Source+of+Mouse+Strain,+and+Regional+Localization.">Functional Specializations of Intestinal Dendritic Cell and Macrophage Subsets That Control Th17 and Regulatory T Cell Responses Are Dependent on the T Cell/APC Ratio, Source of Mouse Strain, and Regional Localization.</a> J. Immunol. , 187-733 (2011).</li><li>Kim, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nod2+sensor+promotes+intestinal+pathogen+eradication+via+the+chemokine+CCL2-dependent+recruitment+of+inflammatory+monocytes.">The Nod2 sensor promotes intestinal pathogen eradication via the chemokine CCL2-dependent recruitment of inflammatory monocytes.</a> Immunity. 34, 769-780 (2011).</li><li>Schulz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+CD103+,+but+not+CX3CR1+,+antigen+sampling+cells+migrate+in+lymph+and+serve+classical+dendritic+cell+functions.">Intestinal CD103+, but not CX3CR1+, antigen sampling cells migrate in lymph and serve classical dendritic cell functions.</a> J. Exp. Med. 206, 3101-3114 (2009).</li><li>Jaensson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+intestinal+CD103++dendritic+cells+display+unique+functional+properties+that+are+conserved+between+mice+and+humans.">Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans.</a> J. Exp. Med. 205, 2139-2149 (2008).</li><li>Uematsu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+humoral+and+cellular+gut+immunity+by+lamina+propria+dendritic+cells+expressing+Toll-like+receptor+5.">Regulation of humoral and cellular gut immunity by lamina propria dendritic cells expressing Toll-like receptor 5.</a> Nat. Immunol. 9, 769-776 (2008).</li><li>Schenk, M., Bouchon, A., Seibold, F., Mueller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TREM-1--expressing+intestinal+macrophages+crucially+amplify+chronic+inflammation+in+experimental+colitis+and+inflammatory+bowel+diseases.">TREM-1--expressing intestinal macrophages crucially amplify chronic inflammation in experimental colitis and inflammatory bowel diseases.</a> J. Clin. Invest. 117, 3097-3106 (2007).</li><li>Sun, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+intestine+lamina+propria+dendritic+cells+promote+de+novo+generation+of+Foxp3+T+reg+cells+via+retinoic+acid.">Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 T reg cells via retinoic acid.</a> J. Exp. Med. 204, 1775-1785 (2007).</li><li>Kamada, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormally+differentiated+subsets+of+intestinal+macrophage+play+a+key+role+in+Th1-dominant+chronic+colitis+through+excess+production+of+IL-12+and+IL-23+in+response+to+bacteria.">Abnormally differentiated subsets of intestinal macrophage play a key role in Th1-dominant chronic colitis through excess production of IL-12 and IL-23 in response to bacteria.</a> J. Immunol. 175, 6900-6908 (2005).</li><li>Denning, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++Th+cells+resembling+regulatory+T+cells+that+inhibit+chronic+colitis+differentiate+in+the+absence+of+interactions+between+CD4+and+class+II+MHC.">CD4+ Th cells resembling regulatory T cells that inhibit chronic colitis differentiate in the absence of interactions between CD4 and class II MHC.</a> J. Immunol. 171, 2279-2286 (2003).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

<img src="/files/ftp_upload/4040/4040fig3.jpg" alt="Figure 3"/> Figure 3. Facteurs importants pour l&#39;optimisation du rendement des cellules et l&#39;expression des antigènes de surface. Rendement cellulaire et l&#39;expression des antigènes de surface sont directement touchés par la durée de la digestion des tissus, les caractéristiques spécifiques de la collagénase, le degré de tissus, de hachage et la présence ou l&#39;absence d&#39;inflammation, ce qui peut affecter l&#39;intégrit?...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> PBS 1X, Ca 2 + - et Mg 2 + sans </td><td></td><td></td><td></td></tr><tr><td> Hank solution saline équilibrée (HBSS) avec le rouge de phénol </td><td> Fisher Scientific </td><td> SH3001603 </td><td></td></tr><tr><td> Le bicarbonate de sodium </td><td> Sigma </td><td> S6014 </td><td></td></tr><tr><td> HEPES 1M en NaCl à 0,85% </td><td> Lonza </td><td> 17-737E </td><td></td></tr><tr><td> Sérum de veau fœtal (FBS) </td><td> Atlanta biologiques </td><td> S11150H </td><td> Inactivé à la chaleur </td></tr><tr><td> EDTA 0,5 M (pH 8,0) </td><td> Cellgro </td><td> 46-034-CI </td><td></td></tr><tr><td> Collagenase type VIII </td><td> Sigma </td><td> C2139 </td><td></td></tr><tr><td> DNase I </td><td> Roche </td><td> 14785000 </td><td> Solution mère: 100mg/ml </td></tr><tr><td> Live / Dead Kit fixable Aqua Dead Cell Stain pour 405 nm d&#39;excitation </td><td> Invitrogen </td><td> L34957 </td><td> Utilisez moins 1:1000 </td></tr><tr><td> CD45-PerCP mAb (30F11) </td><td> BD </td><td> 557235 </td><td> Utilisez au 1:100 </td></tr><tr><td> CD103-PE mAb (M290) </td><td> BD </td><td> 557495 </td><td> Utilisez au 1:100 </td></tr><tr><td> FcyRIII / II mAb (2.4G2) </td><td> BD </td><td> 553141 </td><td> Utilisez au 1:200 </td></tr><tr><td> CD11c-APC mAb (N418) </td><td> eBioscience </td><td> 17-0114-82 </td><td> Utilisez au 1:100 </td></tr><tr><td> CMH-II (I-Ab)-Alexa Fluor 700 mAb </td><td> eBioscience </td><td> 56-5321-82 </td><td> Utilisez au 1:100 </td></tr><tr><td> CD11b-eFluor 450mAb (M1/70) </td><td> eBioscience </td><td> 48-0112-82 </td><td> Utilisez au 1:200 </td></tr><tr><td> F4/80-PE-Cy7 mAb (BM8) </td><td> eBioscience </td><td> 25-4801-82 </td><td></td></tr><tr><td> CD11b microbilles </td><td> Miltenyi Biotec </td><td> 130-049-601 </td><td></td></tr><tr><td> CD11c microbilles </td><td> Miltenyi Biotec </td><td> 130-052-001 </td><td></td></tr><tr><td> Tubes de 50 ml coniques </td><td> BD Falcon </td><td> 352098 </td><td></td></tr><tr><td> Simple filtre en treillis </td><td> Chefmate </td><td></td><td></td></tr><tr><td> Petit bateau pèsent </td><td> Fisher Scientific </td><td> 08-732-116 </td><td></td></tr><tr><td> 100 tamis cellulaire pm </td><td> BD Falcon </td><td> 352360 </td><td></td></tr><tr><td> 40 tamis cellulaire pm </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> 5 ml à fond rond en polystyrène tubes </td><td> BD Falcon </td><td> 352235 </td><td> Utilisez au 1:100 </td></tr><tr><td> MaxQ 4450 paillasse agitateur orbital </td><td> Thermo Scientific </td><td></td><td></td></tr><tr><td> LS MACS colonne </td><td> Miltenyi Biotec </td><td> 130-042-401 </td><td></td></tr><tr><td> LSR II </td><td> BD </td><td></td><td></td></tr><tr><td> FACSAria II </td><td> BD </td><td></td><td></td></tr></tbody></table>

isolation and characterization of dendritic cells and macrophages from the mouse intestine

Dans l&#39;intestin résident les populations uniques de innées et adaptatives des cellules immunitaires qui sont impliqués dans la promotion de la tolérance envers la flore commensale et d&#39;antigènes alimentaires tout en concomitance sur le point restant à monter vers les réponses inflammatoires 1,2 pathogènes invasifs. Cellules présentatrices d&#39;antigènes, en particulier les macrophages, les PED et jouent un rôle crucial dans le maintien de l&#39;homéostasie intestinale immunitaire par leur capacité de détecter et de répondre de façon appropriée le microbiote 3-14. L&#39;isolement efficace des pays en développement intestinales et les macrophages est une étape cruciale pour caractériser le phénotype et la fonction de ces cellules. Alors que de nombreuses méthodes efficaces d&#39;isoler les cellules immunitaires intestinales, y compris les pays en développement et les macrophages, ont été décrits 6,10,15-24, beaucoup comptent sur ​​les temps des digestions longues qui peuvent influencer négativement l&#39;expression des antigènes de surface cellulaire, la viabilité cellulaire, et / ou le rendement des cellules. Ici, nous détaillons une méthode pour l&#39;isolement rapide d&#39;un grand nombre de viable, les PED intestinales et les macrophages. La caractérisation phénotypique des CD intestinales et les macrophages est effectuée par coloration directe isolés cellules intestinales avec spécifiques marquées par fluorescence des anticorps monoclonaux pour l&#39;analyse des flux multi-couleurs cytométrie. En outre, DC de haute pureté et les populations de macrophages sont isolés pour des études fonctionnelles utilisant CD11c et CD11b perles de cellules activées de tri magnétique suivi par le tri cellulaire.

Watch this Scientific Journal Video about Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine at JoVE.com

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

Ici, nous détaillons une méthode pour l&#39;isolement rapide des cellules dendritiques de souris intestinales (DCS) et les macrophages. Caractérisation phénotypique des CD intestinales et les macrophages est effectuée en utilisant plusieurs couleurs d&#39;écoulement analyse cytométrique tandis magnétique enrichissement bourrelet suivie par tri cellulaire est utilisé pour donner des populations de grande pureté pour des études fonctionnelles.

L&#39;isolement et la caractérisation des cellules dendritiques et les macrophages de l&#39;intestin de souris

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

isolation-characterization-dendritic-cells-macrophages-from-mouse

Research

JoVE Journal

Immunology and Infection

38.9K vues.  Emory University. Ici, nous détaillons une méthode pour l'isolement rapide des cellules dendritiques de souris intestinales (DCS) et les macrophages. Caractérisation phénotypique des CD intestinales et les macrophages est effectuée en utilisant plusieurs couleurs d'écoulement analyse cytométrique tandis magnétique enrichissement bourrelet suivie par tri cellulaire est utilisé pour donner des populations de grande pureté pour des études fonctionnelles.

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

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

L&#39;isolement des cellules de souris poumon dendritiques

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

Recherche

Immunologie et infection

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

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

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

Isolement des cellules dendritiques myéloïdes et les cellules épithéliales du thymus humain

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

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

Cellules T Capturez bactéries par Transinfection de cellules dendritiques

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

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 ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Génération et identification de GM-CSF dérivé alvéolaires comme Macrophages et cellules dendritiques à partir de moelle osseuse de souris

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Caractérisation des monocytes dérivées des cellules dendritiques humaines par imagerie cytométrie de flux: une comparaison entre deux protocoles monocytes d&#39;isolement

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Caractérisation phénotypique des macrophages du Rat Kidney par cytométrie de flux

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

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

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

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

Fluorescence-tri cellulaire activé pour la purification de cellules dendritiques plasmacytoïdes de la moelle osseuse de souris

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

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

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

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

Génération de cellules humaines dérivées de monocytes dendritiques du sang total

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

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.