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

Vi takker Aaron Rae (Emory University Department of Pediatrics og børns Healthcare af Atlanta Flow Core) for cellesortering. Dette arbejde blev støttet af NIH tilskud AA01787001, en karriereudvikling Award fra Crohns og Colitis Foundation of America, og en Emory-Egleston Børns Research Center seed-støtte til TLD

1. Dissektion og dissociering af intestinale epitelceller Fremstilling af reagenser og udstyr: <ul><li> Varm Ca2 + / Mg2 +-fri PBS (CMF PBS) til stuetemperatur. </li><li> Varm Ca2 + / Mg2 +-fri HBSS med 5% FBS (CMF HBSS / FBS) og 2 mM EDTA til stuetemperatur. </li><li> Varm orbitalryster til 37 ° C. </li></ul> Bemærk: Trin 1,1 til 1,7 skal udføres så hurtigt som muligt for at minimere omfanget af celle...

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<img src="/files/ftp_upload/4040/4040fig3.jpg" alt="Figur 3"/> Figur 3. Faktorer, der er vigtige for optimering af celle udbytte og overfladeantigen udtryk. Celleudbytte og overfladeantigen udtryk er direkte berørt af varigheden af væv fordøjelsen, de særlige karakteristika kollagenase, graden af væv hakkes og tilstedeværelse eller fravær af inflammation, der kan påvirke vævs integritet og cellularitet. Langvarig vævsfordøjelse kan resultere i nedsat cellelevedygtighed og overfladeantige...

<table border="1"><tbody><tr><td> Navn på reagens </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> 1X PBS, Ca 2 + - og Mg2 +-fri </td><td></td><td></td><td></td></tr><tr><td> Hanks balancerede saltopløsning (HBSS) med phenolrødt </td><td> Fisher Scientific </td><td> SH3001603 </td><td></td></tr><tr><td> Natriumbicarbonat </td><td> Sigma </td><td> S6014 </td><td></td></tr><tr><td> 1M HEPES i 0,85% NaCl </td><td> Lonza </td><td> 17-737E </td><td></td></tr><tr><td> Føtalt bovint serum (FBS) </td><td> Atlanta Biologicals </td><td> S11150H </td><td> Varme-inaktiveret </td></tr><tr><td> 0,5 M EDTA (pH 8,0) </td><td> Cellgro </td><td> 46-034-CI </td><td></td></tr><tr><td> Collagenase typen VIII </td><td> Sigma </td><td> C2139 </td><td></td></tr><tr><td> DNase I </td><td> Roche </td><td> 14785000 </td><td> Stamopløsning: 100mg/ml </td></tr><tr><td> LIVE / DEAD fixable Aqua Dead Cell Stain Kit til 405 nm excitation </td><td> Invitrogen </td><td> L34957 </td><td> Brug på 1:1000 </td></tr><tr><td> CD45-PerCP mAb (30F11) </td><td> BD </td><td> 557235 </td><td> Brug på 1:100 </td></tr><tr><td> CD103-PE mAb (M290) </td><td> BD </td><td> 557495 </td><td> Brug på 1:100 </td></tr><tr><td> FcyRIII / II mAb (2.4G2) </td><td> BD </td><td> 553141 </td><td> Brug på 1:200 </td></tr><tr><td> CD11c-APC mAb (N418) </td><td> eBioscience </td><td> 17-0114-82 </td><td> Brug på 1:100 </td></tr><tr><td> MHC-II (I-Ab)-Alexa Fluor 700 mAb </td><td> eBioscience </td><td> 56-5321-82 </td><td> Brug på 1:100 </td></tr><tr><td> CD11b-eFluor 450mAb (M1/70) </td><td> eBioscience </td><td> 48-0112-82 </td><td> Brug på 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 mikrokugler </td><td> Miltenyi Biotec </td><td> 130-049-601 </td><td></td></tr><tr><td> CD11c mikrokugler </td><td> Miltenyi Biotec </td><td> 130-052-001 </td><td></td></tr><tr><td> 50 ml koniske rør </td><td> BD Falcon </td><td> 352098 </td><td></td></tr><tr><td> Single mesh wire si </td><td> Chefmate </td><td></td><td></td></tr><tr><td> Lille vejer båd </td><td> Fisher Scientific </td><td> 08-732-116 </td><td></td></tr><tr><td> 100 um cellefilter </td><td> BD Falcon </td><td> 352360 </td><td></td></tr><tr><td> 40 um cellefilter </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> 5 ml polystyren rundbundede rør </td><td> BD Falcon </td><td> 352235 </td><td> Brug på 1:100 </td></tr><tr><td> MaxQ 4450 stationære rysteapparat </td><td> Thermo Scientific </td><td></td><td></td></tr><tr><td> LS MACS kolonne </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

Inden for tarmen opholder unikke bestande af medfødte og adaptive immunceller, der er involveret i at fremme tolerance over for kommensale flora og fødevarer antigener, mens samtidig de resterende klar til at montere inflammatoriske reaktioner mod invasive patogener 1,2. Antigenpræsenterende celler, især DC&#39;er og makrofager, spiller kritiske roller i opretholdelsen intestinal immun homøostase via deres evne til sense og passende reagere mikrobiota 3-14. Effektiv isolering af intestinale DC&#39;er og makrofager, er et kritisk trin i karakterisere fænotypen og funktion af disse celler. Mens mange effektive metoder til isolering af intestinale immunceller, herunder DC&#39;er og makrofager, er blevet beskrevet 6,10,15-24 mange stole på lange fordøjelser gange kan negativt påvirke celleoverfladeantigen ekspression, cellelevedygtighed og / eller celleudbytte. Her har vi detaljeret en metode til hurtig isolering af et stort antal viable, intestinale DC&#39;er og makrofager. Fænotypisk karakterisering af intestinale DC&#39;er og makrofager udføres ved direkte farvning isolerede intestinale celler med specifikke fluorescens-mærkede monoklonale antistoffer for flerfarvede flowcytometrisk analyse. Desuden er meget ren DC-og makrofaglinjer populationer isoleret til funktionelle undersøgelser under anvendelse af CD11c og CD11b magnetisk cellesortering perler efterfulgt af cellesortering.

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

Her har vi detalje en metode til hurtig isolering af muse intestinale dendritiske celler (DCS) og makrofager. Fænotypisk karakterisering af intestinale DC&#39;er og makrofager udføres under anvendelse af flerfarvede flowcytometrisk analyse under magnetisk perle tilsætning efterfulgt af cellesortering anvendes til opnåelse af særdeles rene populationer til funktionelle undersøgelser.

Isolering og karakterisering af dendritiske celler og makrofager fra mus Tarmen

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

Research

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Fænotypisk karakterisering af makrofager fra rottenyre ved flowcytometri

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