The overall goal of this method is to isolate gingival immune cells for the in-depth characterization of the immune responses related to oral immunity and periodontitis. This method can help answer key questions in the field of oral immunity, such as what cellular factors are implicated in the pathogenesis of periodontitis. The main advantages of this technique are that it allows the evaluation of the oral immune cell network and the isolation of enough viable cells for downstream in-vitro investigations.
As a first step of this protocol, begin by placing pins to immobilize the body of the mouse and open the thoracic and abdominal cavities to expose the heart and internal organs. Next, make an incision in the vena cava, and use a 3-milliliter syringe, equipped with a 27-gauge needle, to profuse 3 milliliters of PBS via the left ventricle. When all of the blood has been flushed, pin the head of the mouse onto a dissecting pad, with the stomach facing up.
Then use scissors to cut each commissure of the lip towards the neck, separating the skin that covers the mandible and neck region, and exposing the underlying tissues and muscles. Cut between the lower incisors to separate both sides of the mandible. Immobilize each side of the separated mandible with 25-gauge needles to access the oral cavity.
After identifying the palate, use a number 10 surgical blade to make vertical incisions just anterior to the first molar, and posterior to the third molar. Then dissect the tissue away from the nasal cavity, and also dissect the blocks from the mandible. Then 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 one 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 millilters of cold DNase medium, and centrifuge the filtrate.
Then resuspend the palate in 1 milliliter of complete medium, and determine the number of viable cells. The isolation and processing of immune cells as just demonstrated, yields a sufficient number of cells to perfom an ex-vivo stimulation and characterization of the gingival cell cytokine secretion patterns from both wild-type, as well as periodontitis-susceptible animals. Indeed, the murine gingival immune cell composition can be analyzed as demonstrated in these density plots evaluating the t and natural killer cell compartments in periodontitis-susceptible LFA knockout mice.
Using the appropriate gating strategies, the intracellular cytokine production of TCR beta CD4 positive, TCR beta CD8 positive, and TCR gamma delta positive t cells, as well as NK, and innate lymphoid cells, can also be determined. Once mastered, the cells can be isolated in three hours for immunostaining if the technique is performed properly. While attempting this procedure, it's important to remember to complete all of the steps efficiently and quickly, keeping the cells on ice whenever they are not being manipulated.
Following this procedure, other methods, like cell sorting, can be performed to answer additional questions related to the function of the specific cell subsets.
