Isolation of Gingival Immune Cell Network from Murine Teeth Blocks
Transcrição
Eliminate the excess tissue from the border of the gingiva, and transfer the maxillary and mandibular blocks into a 50-milliliter conical tube containing 5 milliliters of collagenase and DNase on ice. Digest the tissue in a shaker incubator at 37 degrees Celsius for 1 hour, adding 50 microliters of 0.5 molar EDTA during the last five minutes. Then, add 5 milliliters of cold DNase medium to the tissues, and gently mix the tube contents by swirling. Next, transfer the four blocks of tissue into a Petri dish, and cover them with 500 microliters of fresh DNase medium.
Use a scalpel to remove the gingiva from each block of tissue and transfer the entire contents of the dish into a 70-micron cell strainer on top of a 50-milliliter tube. Wash the Petri dish and strainer with 3 to 5 milliliters of fresh DNase medium, and then, use the plunger of a sterile 3-milliliter syringe to mash the gingival tissue against the mesh of the strainer. Rinse the strainer with 30 to 35 milliliters of cold DNase medium, and centrifuge the filtrate. Then, resuspend the pellet in one milliliter of complete medium, and determine the number of viable cells.
