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

Wij danken Aaron Rae (Emory University Department of Pediatrics en de Children&#39;s Healthcare van Atlanta Flow Core) voor celsortering. Dit werk werd ondersteund door NIH-subsidie ​​AA01787001, een Career Development Award van de Crohn en Colitis Foundation of America, en een Emory-Egleston Children&#39;s Research Center zaad subsidie ​​TLD

1. Dissectie en Dissociatie van darmepitheelcellen Voorbereiding van de reagentia en apparatuur: <ul><li> Warm Ca2 + / Mg2 +-vrij PBS (CMF PBS) tot kamertemperatuur. </li><li> Warm Ca2 + / Mg2 +-vrij HBSS met 5% FBS (CMF HBSS / FBS) en 2 mM EDTA tot kamertemperatuur. </li><li> Warm Orbital Shaker tot 37 ° C. </li></ul> Opmerking: Stap 1,1 tot 1,7 worden uitgevoerd zo snel mogelijk de mate van celdood te m...

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<img src="/files/ftp_upload/4040/4040fig3.jpg" alt="Figuur 3"/> Figuur 3. Factoren die belangrijk zijn voor het optimaliseren van cel-opbrengst en oppervlakte-antigeen expressie. Cell opbrengst en oppervlakte-antigeen expressie worden rechtstreeks beïnvloed door de duur van weefsel de spijsvertering, de specifieke kenmerken van de collagenase, de mate van weefsel hakken, en de aanwezigheid of afwezigheid van ontsteking, die van invloed kunnen weefsel integriteit en cellulariteit. Langdurige weefse...

<table border="1"><tbody><tr><td> Naam van het serum </td><td> Vennootschap </td><td> Catalogusnummer </td><td> Reacties </td></tr><tr><td> 1X PBS, Ca2 + - en Mg + 2 vrij </td><td></td><td></td><td></td></tr><tr><td> Balanced Salt Hank&#39;s oplossing (HBSS) met fenol rood </td><td> Fisher Scientific </td><td> SH3001603 </td><td></td></tr><tr><td> Natriumbicarbonaat </td><td> Sigma </td><td> S6014 </td><td></td></tr><tr><td> 1M HEPES in 0,85% NaCl </td><td> Lonza </td><td> 17-737E </td><td></td></tr><tr><td> Foetaal runderserum (FBS) </td><td> Atlanta biologicals </td><td> S11150H </td><td> Warmte geïnactiveerd </td></tr><tr><td> 0,5 M EDTA (pH 8,0) </td><td> Cellgro </td><td> 46 tot 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> Stock oplossing: 100mg/ml </td></tr><tr><td> Live / Dead Muur Aqua Dead Cell Stain Kit voor 405 nm excitatie </td><td> Invitrogen </td><td> L34957 </td><td> Gebruik op 1:1000 </td></tr><tr><td> CD45-PerCP mAb (30F11) </td><td> BD </td><td> 557235 </td><td> Gebruik op 1:100 </td></tr><tr><td> CD103-PE mAb (M290) </td><td> BD </td><td> 557495 </td><td> Gebruik op 1:100 </td></tr><tr><td> FcγRIII / II mAb (2.4G2) </td><td> BD </td><td> 553141 </td><td> Gebruik op 1:200 </td></tr><tr><td> CD11c-APC mAb (N418) </td><td> eBioscience </td><td> 17-0114-82 </td><td> Gebruik op 1:100 </td></tr><tr><td> MHC-II (I-Ab)-Alexa Fluor 700 mAb </td><td> eBioscience </td><td> 56-5321-82 </td><td> Gebruik op 1:100 </td></tr><tr><td> CD11b-eFluor 450mAb (M1/70) </td><td> eBioscience </td><td> 48-0112-82 </td><td> Gebruik op 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 microkorrels </td><td> Miltenyi Biotec </td><td> 130-049-601 </td><td></td></tr><tr><td> CD11c microkorrels </td><td> Miltenyi Biotec </td><td> 130-052-001 </td><td></td></tr><tr><td> 50 ml conisch </td><td> BD Falcon </td><td> 352098 </td><td></td></tr><tr><td> Single gaas zeef </td><td> Chefmate </td><td></td><td></td></tr><tr><td> Kleine wegen boot </td><td> Fisher Scientific </td><td> 08-732-116 </td><td></td></tr><tr><td> 100 um zeef cel </td><td> BD Falcon </td><td> 352360 </td><td></td></tr><tr><td> 40 urn zeef cel </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> 5 ml polystyreen ronde bodem buizen </td><td> BD Falcon </td><td> 352235 </td><td> Gebruik op 1:100 </td></tr><tr><td> MaxQ 4450 benchtop schudapparaat </td><td> Thermo Scientific </td><td></td><td></td></tr><tr><td> LS MACS kolom </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

Binnen de darm verblijven, unieke populaties van aangeboren en adaptieve immuuncellen die betrokken zijn bij het ​​bevorderen van tolerantie ten opzichte van commensale flora en voedsel antigenen, terwijl gelijktijdig de resterende klaar om ontstekingsreacties te monteren in de richting van invasieve pathogenen 1,2. Antigeen presenterende cellen, in het bijzonder DCs en macrofagen, spelen een cruciale rol bij het ​​handhaven van intestinale immuun homeostase via hun vermogen om betekenis en adequaat te reageren op de microbiota 3-14. Efficiënte isolatie van intestinale DCs en macrofagen is een cruciale stap in het karakteriseren van het fenotype en functie van deze cellen. Hoewel veel effectieve methoden voor het isoleren intestinale immuuncellen, zoals DCs en macrofagen, zijn beschreven 6,10,15-24 veel vertrouwen op lange digesties maal negatief kan beïnvloeden cel oppervlakte antigen expressie levensvatbaarheid van de cellen en / of cel opbrengst. Hier hebben we details van een methodologie voor de snelle isolatie van grote aantallen viable-, darm-DCs en macrofagen. Fenotypische karakterisatie van intestinale DCs en macrofagen wordt uitgevoerd door het rechtstreeks kleuring geïsoleerde darmcellen met fluorescentie-gelabelde monoklonale antilichamen voor meerkleuren flowcytometrie. Bovendien zijn zeer zuiver DC populaties en macrofaag geïsoleerd functionele onderzoeken waarin CD11c en CD11b magnetische geactiveerde celsortering kralen gevolgd door celsortering.

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

Hier hebben we details van een methodologie voor de snelle isolatie van de muis intestinale dendritische cellen (DC&#39;s) en macrofagen. Fenotypische karakterisering van intestinale DCs en macrofagen wordt uitgevoerd met behulp van multi-kleuren flowcytometrische analyse, terwijl magnetische kraal verrijking gevolgd door celsortering wordt gebruikt om zeer zuivere populaties voor functionele studies opleveren.

Isolatie en karakterisering van dendritische cellen en macrofagen van de muis Darm

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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