Name: isolation and characterization of dendritic cells and macrophages from the mouse intestine
Uploaded: 2012-05-21T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine at JoVE.com

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 êtr...

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<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 ...

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