Isolering og karakterisering af dendritiske celler og makrofager fra mus Tarmen
Summary
Her har vi detalje en metode til hurtig isolering af muse intestinale dendritiske celler (DCS) og makrofager. Fænotypisk karakterisering af intestinale DC'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.
Abstract
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'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'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'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'er og makrofager. Fænotypisk karakterisering af intestinale DC'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.
Protocol
Discussion
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
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
Materials
| Name of the Reagent | Company | Catalogue number | Comments |
| 1X PBS, Ca2+– and Mg2+-free | |||
| Hank’s balanced salt solution (HBSS) with phenol red | Fisher Scientific | SH3001603 | |
| Sodium bicarbonate | Sigma | S6014 | |
| 1M HEPES in 0.85% NaCl | Lonza | 17-737E | |
| Fetal bovine serum (FBS) | Atlanta biologicals | S11150H | Heat-inactivated |
| 0.5M EDTA (pH 8.0) | Cellgro | 46-034-CI | |
| Collagenase type VIII | Sigma | C2139 | |
| DNase I | Roche | 14785000 | Stock solution: 100mg/ml |
| LIVE/DEAD Fixable Aqua Dead Cell Stain Kit for 405 nm excitation | Invitrogen | L34957 | Use at 1:1000 |
| CD45-PerCP mAb (30F11) | BD | 557235 | Use at 1:100 |
| CD103-PE mAb (M290) | BD | 557495 | Use at 1:100 |
| FcγRIII/II mAb (2.4G2) | BD | 553141 | Use at 1:200 |
| CD11c-APC mAb (N418) | eBioscience | 17-0114-82 | Use at 1:100 |
| MHC-II (I-Ab)-Alexa Fluor 700 mAb | eBioscience | 56-5321-82 | Use at 1:100 |
| CD11b-eFluor 450 mAb (M1/70) | eBioscience | 48-0112-82 | Use at 1:200 |
| F4/80-PE-Cy7 mAb (BM8) | eBioscience | 25-4801-82 | |
| CD11b microbeads | Miltenyi Biotec | 130-049-601 | |
| CD11c microbeads | Miltenyi Biotec | 130-052-001 | |
| 50 mL conical tubes | BD Falcon | 352098 | |
| Single mesh wire strainer | Chefmate | ||
| Small weigh boat | Fisher Scientific | 08-732-116 | |
| 100 μm cell strainer | BD Falcon | 352360 | |
| 40 μm cell strainer | BD Falcon | 352340 | |
| 5 mL polystyrene round-bottom tubes | BD Falcon | 352235 | Use at 1:100 |
| MaxQ 4450 benchtop orbital shaker | Thermo Scientific | ||
| LS MACS column | Miltenyi Biotec | 130-042-401 | |
| LSR II | BD | ||
| FACSAria II | BD |
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