Isolation of Gingival Immune Cell Network from Murine Teeth Blocks
DEŞİFRE METNİ
The gingiva, or gum tissue, surrounds the cervical portion of teeth and the jawbone.
Gingival tissue comprises an epithelium layer, separated from the underlying connective tissue by a collagen-IV-rich basement membrane. The gingival connective tissue contains an immune cell network, including lymphocytes, mast cells, and neutrophils that protect the gingiva from infections.
To isolate the gingival immune cell network, take murine teeth blocks containing firmly attached gingival tissue. Treat the blocks with an enzyme cocktail — containing type four collagenases and DNases.
Collagenases enter the tissue, degrade collagen-IV, and disrupt the basement membrane and gingival epithelium, causing the gingiva to loosen from the teeth. Meanwhile, DNases degrade the DNA contaminants. Add a chelating agent to the tube; to remove divalent cations and prevent cell aggregation.
Transfer the digested teeth blocks to a petri dish containing DNase medium. Extract the gingiva tissue from the teeth blocks, and transfer it into a cell strainer.
Using a plunger, mash the tissue. This mechanical dissociation disrupts the gingival connective layer, releasing the immune cells from the tissue. Wash the strainer with media and collect the released cells in the filtrate.
Centrifuge the filtrate, pelletizing the cells, and discard the supernatant containing residual enzymes and debris. Resuspend the cell population carrying immune cells in complete media, ready for future analysis.
