Vi har etableret en teknik til isolering, fænotypiske karakterisering og funktionel analyse af immunceller fra murine gingiva.
Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.
Gingivale væv omgiver human og murin tandsæt og er konstant udsat for den komplekse biofilm af tanden 1. Immuncellen netværk politiarbejde gingival barriere er afgørende for at bevare vævsintegritet, sikrer homøostase med de lokale kommensale mikrober og, på samme tid, hvilket giver en effektiv immunitet mod patogen udfordring 2. For at opnå homeostase, er immunsystemet omhyggeligt skræddersyet til den gingival miljø skaber en højt specialiseret immun celle netværk, men lille detalje er kendt af de gingivale immun cellepopulationer og deres rolle i at opretholde væv immunitet 2.
Når immun homøostase er forstyrret på gingiva, enten gennem øget vært modtagelighed og / eller tilstedeværelse af dysbiotic mikrobielle samfund, en inflammatorisk tilstand, opstår parodontitis 3 4. Parodontitis er en almindelig inflammatorisk sygdom, der fører til tab af tand sTØTTE strukturer. I sine alvorlige former er det set hos ca. 10% af den almindelige befolkning 5. Dissekere de centrale faktorer involveret i parodontitis modtagelighed og progression har vist sig vanskeligt 6. Imidlertid har dyremodeller været yderst nyttige i at forstå mekanismerne i parodontitis initiering og progression 7. Modeller kan anvendes til at definere de centrale cellepopulationer og molekylære mediatorer, der er afgørende for at opretholde immunforsvaret homeostase og drive udviklingen af parodontitis. En sådan indsigt vil forvandle vores forståelse af gingiva-specifik styring af immun homøostase og fremme vores nuværende forståelse af sygdommens patogenese.
Den nuværende teknik med held giver et stort antal af immunceller (fra en enkelt mus) passer, ikke kun for fænotypisk karakterisering, men også for funktionelle studier ex vivo. En anden gruppe havde tidligere offentliggjort en protokol at isolere og karakterisere murine gingival immunceller og indført værdien af at anvende flerfarvet flowcytometri i studiet af parodontitis i dyremodeller 9. Efter betydelig fejlfinding nøglen modifikation i denne protokol var at omfatte dissektion af hele blokk…
The authors have nothing to disclose.
Authors were funded in part by the intramural program of NIDCR (N.M.M) and supported by a Wellcome Trust Stepping Stones Fellowship (097820/Z/11/B to J.E.K) and by a Manchester Collaborative Centre for Inflammation Research grant (to J.E.K). The authors thank Teresa Wild for critically reviewing the manuscript.
Fine Scissors | Fine science tools | 14058-11 | |
Scalpel Handle #3 | Fine science tools | 10003-12 | |
Scalpel Blades #10 | Fine science tools | 10010-00 | Sterile |
Splinter Forceps | Integra Miltex | 6-304 | |
Needles with regular bevel | BD Medical | 305109 | 27G, 12.7 mm length |
Monoject syringes | Covidien | 8881513934 | Luer-lock tip, 3mL |
PBS, pH 7.4 | Life Technologies | 10010-049 | Without Calcium and Magnesium |
RPMI 1640 | Lonza | 12-167F | Without L-glutamine |
DNase I from bovine pancreas | Sigma-Aldrich | DN25-1G | |
Collagenase type IV | Gibco (by Life technologies) | 17104-019 | |
Fetal Bovine Serum | Gemini Bio-products | 100-106 | |
Gentamicin 50 mg/ml | Quality biological | 120-098-661EA | |
Pen Strep | Gibco (by Life technologies) | 15140-122 | |
L-Glutamine | Gibco (by Life technologies) | 25030-081 | |
0.5M EDTA pH 8.0 | Quality biological | 351-027-721EA | |
50 mL tubes | Corning | 352070 | Polypropylene, sterile |
70 μM Cell Strainers | Corning | 352350 | |
Petri dishes | Corning | 351029 | Sterile |
5 mL FACS tubes | Corning | 352052 | Sterile |
BD GolgiPlug | BD Biosciences | 555029 | Contains brefeldin A solution |
Phorbol 12-Myristate 13-Acetate (PMA) | Sigma-Aldrich | P8139 | |
Ionomycin Calcium Salt | Sigma-Aldrich | 13909 | |
Saponin from quillaja bark | Sigma-Aldrich | S4521 | |
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit | Life Technologies | L34957 | |
Anti-Mouse CD45 Alexa Fluor 700 | eBioscience | 56-0451-82 | |
Anti-Mouse CD4 eFluor 450 | eBioscience | 48-0042-82 | |
Anti-Mouse TCR beta APC eFluor 780 | eBioscience | 47-5961-82 | |
Anti-Mouse gamma delta TCR FITC | eBioscience | 11-5711-82 | |
Anti-Mouse IL-17A APC | eBioscience | 12-7311-82 | |
Anti-Mouse IFN-γ PE | eBioscience | 11-5931-82 | |
Anti-Mouse NK1.1 PE-Cy7 | eBioscience | 25-5941-82 | |
Anti-Mouse CD90.2 APC eFluor 780 | eBioscience | 47-0902-82 | |
Anti-Mouse CD3e FITC | eBioscience | 11-0031-82 | |
Anti-Mouse CD19 FITC | eBioscience | 11-0193-82 | |
Anti-Mouse CD11b FITC | eBioscience | 11-0112-82 | |
Anti-Mouse CD11c FITC | eBioscience | 11-0114-82 | |
Anti-Mouse TCR beta FITC | eBioscience | 11-5961-82 | |
Anti-Mouse Ly-6G FITC | eBioscience | 11-5931-82 | |
Anti-Mouse Ly-6C FITC | BD Pharmingen | 553104 |