Isolering og karakterisering av dendrittiske celler og makrofager fra musen Intestine
Summary
Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.
Abstract
Innenfor tarmen bor unike bestander av medfødte og ervervede immun celler som er involvert i å fremme toleranse overfor Commensal flora og mat antigener mens samtidig resterende klar til å montere inflammatoriske responser mot invasiv patogener 1,2. Antigenpresenterende celler, særlig DCS og makrofager, spiller avgjørende roller i å opprettholde intestinal immune homeostase via deres evne til å oppfatte og å reagere på den bakterieflora 3-14. Effektiv isolering av tarm DCS og makrofager er et kritisk punkt i karakterisere fenotypen og funksjon av disse cellene. Mens mange effektive metoder for å isolere intestinal immunceller, blant annet DCS og makrofager, har blitt beskrevet 6,10,15-24, mange er avhengige av lange digestions tider som kan negativt påvirke celleoverflateantigen uttrykk, celleviabilitet, og / eller celle yield. Her detalj vi en metode for rask isolering av et stort antall viable, intestinal DCS og makrofager. Fenotypisk karakterisering av intestinale DCS og makrofager blir utført av direkte flekker isolerte tarmcellene med konkrete fluorescens-merkede monoklonale antistoffer for multi-farge flowcytometrisk analyse. Videre er svært ren DC og macrophage populasjoner isolert for funksjonelle studier utnytte CD11c og CD11b magnetisk aktivert celle sortering perler etterfulgt av celle sortering.
Protocol
Discussion
Figur 3. Faktorer som er viktige for optimalisering av cellen yield og overflateantigen uttrykk. Cell yield og overflateantigen uttrykk er direkte berørt av varigheten av vev fordøyelse, de spesifikke egenskapene til collagenase, grad av vev hakking, og tilstedeværelse eller fravær av betennelse, som kan påvirke vev integritet og cellularitet. Langvarig vev fordøyelsen kan resultere i redusert celleviabilitet og overflateantigen u…
Declarações
The authors have nothing to disclose.
Acknowledgements
Vi takker Aaron Rae (Emory University Department of Pediatrics og Barnas Healthcare of Atlanta Flow Core) for celle sortering. Dette arbeidet ble støttet av NIH AA01787001 bevilgning, en Career Development Award fra Crohns og kolitt Foundation of America, og en Emory-Egleston Barnas Research Center frø stipend 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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