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 tackar Aaron Rae (Emory University Institutionen för pediatrik och barn Healthcare Atlanta Flow Core) för cell sortering. Detta arbete stöddes av NIH bidrag AA01787001, en Career Development Award från Crohns och kolit Foundation of America, och en Emory-Egleston barn Research Center säd bidrag till TLD

1. Dissektion och Dissociation av tarmepitelceller Beredning av reagens och utrustning: <ul><li> Varm Ca 2 + / Mg2 +-fri PBS (CMF PBS) till rumstemperatur. </li><li> Varm Ca 2 + / Mg2 +-fri HBSS med 5% FBS (CMF HBSS / FBS) och 2 mM EDTA till rumstemperatur. </li><li> Varm orbitalskakare till 37 ° C. </li></ul> Notera: Stegen 1,1 till 1,7 måste utföras så snabbt som möjligt för att minimera omfattning...

<ol>
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<img src="/files/ftp_upload/4040/4040fig3.jpg" alt="Figur 3"/> Figur 3. Faktorer som är viktiga för optimering av cell avkastning och ytantigen uttryck. Cell avkastning och ytantigen uttryck direkt påverkas av varaktigheten av vävnad matsmältningen de särskilda egenskaper kollagenas, graden av vävnaden mals och förekomsten eller frånvaron av inflammation, som kan påverka vävnader integritet och cellularitet. Långvarig vävnaden matsmältningen kan resultera i minskad cellviabilitet och ...

<table border="1"><tbody><tr><td> Namn på Reagent </td><td> Företaget </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> 1 X PBS, Ca 2 + - och Mg2 +-fri </td><td></td><td></td><td></td></tr><tr><td> Hanks balanserade saltlösning (HBSS) med fenolrött </td><td> Fisher Scientific </td><td> SH3001603 </td><td></td></tr><tr><td> Natriumbikarbonat </td><td> Sigma </td><td> S6014 </td><td></td></tr><tr><td> 1 M HEPES i 0,85% NaCl </td><td> Lonza </td><td> 17-737E </td><td></td></tr><tr><td> Fetalt bovint serum (FBS) </td><td> Atlanta Biologicals </td><td> S11150H </td><td> Värmeinaktiverat </td></tr><tr><td> 0,5 M EDTA (pH 8,0) </td><td> Cellgro </td><td> 46-034-Cl </td><td></td></tr><tr><td> Collagenase typ VIII </td><td> Sigma </td><td> C2139 </td><td></td></tr><tr><td> DNas I </td><td> Roche </td><td> 14785000 </td><td> Stamlösning: 100mg/ml </td></tr><tr><td> LIVE / DEAD torrt magnetpulver, fixerbart Aqua Dead Cell Stain Kit för 405 nm excitation </td><td> Invitrogen </td><td> L34957 </td><td> Användning på 1:1000 </td></tr><tr><td> CD45-PerCP-mAb (30F11) </td><td> BD </td><td> 557.235 </td><td> Använd vid 1:100 </td></tr><tr><td> CD103-PE-mAb (M290) </td><td> BD </td><td> 557.495 </td><td> Använd vid 1:100 </td></tr><tr><td> FcyRIII / II-mAb (2.4G2) </td><td> BD </td><td> 553.141 </td><td> Användning på 1:200 </td></tr><tr><td> CD11c-APC-mAb (N418) </td><td> eBioscience </td><td> 17-0114-82 </td><td> Använd vid 1:100 </td></tr><tr><td> MHC-II (I-Ab)-Alexa Fluor 700-mAb </td><td> eBioscience </td><td> 56-5321-82 </td><td> Använd vid 1:100 </td></tr><tr><td> CD11b-eFluor 450mAb (M1/70) </td><td> eBioscience </td><td> 48-0112-82 </td><td> Användning 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 mikropärlor </td><td> Miltenyi Biotec </td><td> 130-049-601 </td><td></td></tr><tr><td> CD11c mikropärlor </td><td> Miltenyi Biotec </td><td> 130-052-001 </td><td></td></tr><tr><td> 50 ml koniska rör </td><td> BD Falcon </td><td> 352.098 </td><td></td></tr><tr><td> Enda nät trådsil </td><td> Chefmate </td><td></td><td></td></tr><tr><td> Liten väger båten </td><td> Fisher Scientific </td><td> 08-732-116 </td><td></td></tr><tr><td> 100 | im cellfilter </td><td> BD Falcon </td><td> 352.360 </td><td></td></tr><tr><td> 40 | im cellfilter </td><td> BD Falcon </td><td> 35.2340 </td><td></td></tr><tr><td> 5 ml polystyren rundbottnade rör </td><td> BD Falcon </td><td> 352.235 </td><td> Använd vid 1:100 </td></tr><tr><td> MaxQ 4450 bordsmodell orbitalskakare </td><td> Thermo Scientific </td><td></td><td></td></tr><tr><td> LS MACS kolonn </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

Inom tarmen bor unika populationer av medfödda och adaptiva immunceller som är involverade i främjandet av tolerans mot kommensalism flora och antigener mat medan samtidigt resterande redo att montera inflammatoriska reaktioner mot invasiv patogener 1,2. Antigenpresenterande celler, i synnerhet DCS och makrofager, spela kritiska roller i att upprätthålla intestinal immunhomeostas via deras förmåga att känna av och på lämpligt sätt svara på mikrobiota 3-14. Effektiv isolering av intestinala DC och makrofager är ett kritiskt steg vid karakterisering av fenotyp och funktion hos dessa celler. Medan många effektiva metoder för att isolera intestinala immunceller, inklusive DC och makrofager, har beskrivits 6,10,15-24 många förlitar sig på långa digestioner gånger negativt kan påverka cellens uttryck ytantigen, cellviabilitet och / eller cell avkastning. Här, i detalj vi en metod för snabb isolering av ett stort antal viable, intestinala DC och makrofager. Fenotypisk karakterisering av intestinala DC och makrofager sker genom direkt färgning isolerade intestinala celler med specifika fluorescens-märkta monoklonala antikroppar för flerfärgs-flödescytometrisk analys. Dessutom är mycket ren DC och populationer makrofag isolerades för funktionella studier som använder CD11c och CD11b magnetiska cellsortering pärlor följt av cellsortering.

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

Här har vi detalj en metod för snabb isolering av mus-intestinala dendritiska celler (DC) och makrofager. Fenotypisk karakterisering av intestinala DC och makrofager utförs med hjälp av multi-color flödescytometrianalys medan magnetiska pärlor anrikning följt av cellsortering används för att ge mycket rena populationer för funktionella studier.

Isolering och karakterisering av dendritiska celler och makrofager från mus tarm

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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Fluorescensaktiverad cellsortering för Rening av Plasmacytoid dendritiska celler från benmärg från mus

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