Immunology and Infection
Published: July 2nd, 2013
This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.
We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.
Gingiva is the soft tissue surrounding the cervical portion of the teeth and covers the alveolar process (Figure 1). The gingiva is a type of masticatory mucosa that can be further separated into mucosal epithelium and connective tissue (also known as submucosa or lamina propria). The anatomical structure of the gingiva and adjacent teeth allows bacteria to reside and develop plaque (biofilm) that constantly challenges the local immune system. As a result, inflammatory response develops in the gingiva, which in certain circumstances becomes destructive - a condition termed periodontal diseases 2. Basically, plaque-induced periodontal pathol....
Prepare in advance:
1. Upper Gingival Excision Technique
Examples of flow cytometry analysis on gingival cells are presented. Gingival cells pooled from 2 mice were run in an LSR II flow cytometer and analyzed using FlowJo software. Figure 3A demonstrated the distribution of gingival cells from naïve mice in a side scatter (SSC) versus forward scatter (FSC) plot. Gating strategy to identify (i) lymphocytes (ii) monocytes / dendritic cells and (iii) granulocytes is indicated. For comparison purpose, we also present a FACS plot illustrating gingival cells p.......
Maxilla gingival tissues obtained from a single mouse are sufficient for analyzing sub-populations of T and B lymphocytes, as well as their capability to express extracellular and intracellular molecules as we previously described 1. Nevertheless, if rare cell populations are of interest (for example DCs), it is recommended to pool tissues from 2-3 mice. Of note, if preferable, it is possible to peel both the palatal and gingival tissues and then to excise the gingiva (a modification of step 1.8-1.9). It shoul.......
This research was supported by grants from the Israel Science Foundation (No. 1418/11) to AHH and (No. 1933/12) to AW, the German Israeli Foundation for young investigators (GIF Young) to AHH, and the Dr. I. Cabakoff Research Endowment Fund at the Hebrew University-Hadassah School of Dental Medicine to AHH and AW.....
|Name of the reagent
|Worthington Biochemical Corp.
|Collagenase Type II
|Fetal Bovine Serum
|5 ml Polystyrene Round-Bottom Tube
|Cell Strainer 70 μm
|Tissue Culture Dish 35×10 mm
|Anti-mouse CD11c antibody
|Anti-mouse CD45.2 antibody
|Anti-mouse CD4 antibody
|Anti-mouse CD8a antibody
|Anti-mouse CD3 antibody
|Anti-mouse CD326 (Ep-CAM) antibody
|Anti-mouse CD207 (Langerin) antibody
|Anti-mouse I-Ad (MHC-II) antobody
|LSR II Flow Cytometer
|FlowJo Software v 7.6.5
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