Isolation of Gingival Immune Cell Network from Murine Teeth Blocks

Abstract

Source: Dutzan, N. et al., Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network. J. Vis. Exp. (2016)

This video demonstrates the isolation procedure of the gingival immune cell network from a mouse model. The enzymatic digestion releases the gingival tissue from the attached teeth and bone, which is further mechanically digested, releasing immune cells from the gingiva. The isolated cells are ready for immunological experiments.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Prepare in Advance Prepare Complete media: RPMI supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 ug/ml streptomycin, and 10% FBS. Prepare DNase media: 50 ml of RPMI media supplemented with 7.5 µg DNase (Make fresh, keep on ice at all times). Prepare Collagenase-DNase media: 5 ml …

Representative Results

Figure 1: Isolation of murine gingiva. Illustration demonstrating the borders of dissection in the maxilla and the isolated gingival segment to be used for gingival tissue dissection. (B) Segment of the murine maxilla (molar area) with an outline for block dissection, and isolated block. Hematoxylin and Eosin stain (H&E) of a maxillary molar segment with gingival tissues outlined is shown in low (C) and higher magnifica…

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