分离，鉴定和牙龈免疫细胞网络的功能性考试
Summary
我们已经建立了隔离，表型特征，并从小鼠牙龈免疫细胞的功能分析的技术。
Abstract
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.
Introduction
牙龈组织包围人和鼠牙列和经常暴露于齿1的复杂生物膜。免疫细胞网络监管龈屏障对于维持组织完整性，确保与本地共生微生物稳态和，在相同的时间，提供对致病性挑战2有效免疫至关重要。实现动态平衡，免疫系统被仔细针对牙龈环境中创建一个高度专业化的免疫细胞网络，但小细节是已知的齿龈免疫细胞群及其在维持组织免疫2的作用。
当免疫稳态是在齿龈打乱，无论是通过增加宿主易感性和/或dysbiotic微生物群落，炎性病症的存在，牙周炎产生3 4。牙周炎是一种常见的炎性疾病，导致牙齿的S损失95的支持结构。在它的严重的形式，可以看出在一般人群5的约10％。解剖参与牙周炎的易感性和进展的关键因素已被证明难以6。然而，动物模型已经在理解牙周炎起始和进展7的机制是非常有用的。模型可以被用来定义密钥的细胞群体和分子介质是用于维持免疫稳态和牙周炎的驱动发展至关重要。这样的洞察力将改变我们的免疫稳态的具体牙龈调控的了解，进一步我们目前的发病机制的认识。
Protocol
Representative Results
Discussion
当前技术成功地产生合适的大量免疫细胞（从单个小鼠），不仅对表型特征，也用于功能研究离体 。另一组先前公布的协议，以分离和表征鼠牙龈免疫细胞和导入用多色牙周炎的动物模型9的研究流式细胞仪的值。经大量排除在本协议中的关键改变是包括组织的全块解剖，以包括牙龈领区和齿间牙龈（图1）。与单纯的牙龈切除，常全层皮瓣没有实现，而且牙龈的部分尤…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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 |
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