Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

The overall goal of the following method is to analyze the functions of mast cells in vivo using mast cell knockin mice. This is achieved by first generating bone marrow derived cultured mast cells, or BMC MCs or engraftment into mast cell deficient mice. In the second step, the BMC MCs are adoptively transferred into various anatomical sites in the mast cell deficient mice to generate mast cell knockin mice.

Ultimately, the numbers and anatomic distribution of the adoptively transferred mast cells are assessed by flow cytometry and immunohistochemical analysis to evaluate to what extent the engrafted populations resemble those found in wild type mice. The main advantage of this technique that it permits direct assessment of the functions of the engrafted wall type or genetically modified MA cell populations in vivo. This method therefore can help answer key questions in the field of MA cell biology, such as to what extent MA cells or their products can contribute to features of mouse models of physiological, immunological, or pathological processes, including models of human disease.

Generally, individuals learning this method may struggle because mast cell engraft and deficiency can vary depending on the method used to generate BM CMCs, the root of injection, the numbers of BMC MCs injected and the number of weeks one weight after engraftment before performing in vivo experiments. Visual demonstration of this method is critical as aspects of the engraftment procedure are difficult to learn unless one sees exactly how they are performed. To extract the bone marrow first, use sterile dissection tools to harvest the femur, tibia, and fibula from wild type mice without cussing any bone epiphysis.

As each bone is dissected, secure it with forceps and scrape off all of the extraneous tissue. Then place the bone in PBS on ice when all of the bones have been collected, transfer em into a sterile tissue culture hood, then one at a time, secure each bone with forceps and cut off both epiphyses to expose the medullary cavities. Next, using three milliliter syringes, equipped with 30 gauge needles and flushing cold medium, all the red marrow from each bone into a new Petri dish When all of the bones have been flushed, use the same syringe to dissociate the bone marrow cell clusters by repeated gentle aspiration and ejection.

Then transfer the cell suspension into a 15 milliliter centrifuge tube. Fill the tube with cold flushing medium and centrifuge the cells resus. Suspend the cell pellet in 10 milliliters of culture medium.

Then transfer the bone marrow cells into an appropriately sized tissue culture flask. After one to two days of culture, transfer the non-adherence cells to a new flask along with 10 milliliters of fresh culture, medium per mouse during the following weeks. Feed the cells every three to four days is just demonstrated.

Transferring the non-adherence cells to a new flask once a week until no adherence cells are left for intradermal engraftment into the ear pinner Resus. Suspend the BMC MCs at four times 10 to the seven cells per milliliter in cold DMEM. Then aspirate the cells into a one milliliter syringe equipped with a 30 gauge needle and place the syringe on ice.

Next, confirm the appropriate depth of anesthesia by a top pinch of a four to six week old mast cell deficient recipient mouse and apply eye ointment to inject the cells. Use the index finger to expose and stretch the ventral face of the ear pinner, creating vertical pressure on the dorsal face of the ear. Then administer 1 25 microliter intradermal injection of one times 10 to the six BMC MCs into the middle of the ear, and a second 25 microliter injection of the same toward the tip of the pinner.

For intraperitoneal, engraftment resuspend the BMC MCs at one times 10 to the seven cells per milliliter in cold DMEM. Then use a one milliliter syringe equipped with a 25 gauge needle to inject two times 10 to the six BM CMCs in 200 microliters of medium into the peritoneal cavity of the mast cell deficient recipients for intravenous engraftment. Resuspend the BM CMCs at 2.5 times 10 to the seven cells per milliliter in cold DMEM, and then inject five times 10 to the six BMC MCs in 200 microliters of medium via the tail vein.

Four to six weeks after an intraperitoneal engraftment, use a five milliliter syringe equipped with a 25 gauge needle to inject five milliliters of cold PBS into the peritoneal cavity of the recipient animal. Massage the abdomen for 20 seconds to harvest the peritoneal cells. Then use a five milliliter syringe equipped with a 22 gauge needle to slowly aspirate the peritoneal lavage.

The mast cell percentage and total cell number in the peritoneal lavage fluid can then be evaluated by flow cytometry or histo chemistry. To evaluate the mast cells in mesenteric windows, cut open the peritoneal membrane to expose the intestinal tract. Arrange four to five mesenteric windows per mouse onto a slide followed by a one hour fixation in car noise solution.

Add room temperature at the end of the incubation, air dry the slides and remove the intestine. The fixed mesenteric windows will remain attached to the slides. Finally, stain the preparations for 20 minutes at room temperature with chaba staining solution represent flow cytometric analysis and odine blue staining of BM CMCs after one 15 and 45 days of culture In DMEM, supplemented with IL three are shown BM CMCs cultured for 45 days and 95%pure and contain high numbers of cytoplasmic granules.

Therefore, cells from these cultures are suitable for engraftment experiments, whereas cells cultured for 15 days are not in these images. Successful engraftment of the ear penny four weeks after intradermal, engraftment mesenteric windows, the peritoneal cavity six weeks after intraperitoneal engraftment and the lung 12 weeks after intravenous engraftment are shown as expected. Mast cells are present in the tissue samples isolated from wild type mice, whereas no mast cells are detected in the mast cell deficient animals in the mast cell knockin mice.

However, the adoptively transferred BMC MCs appear in similar locations to those observed in the wild type animals. While attempting this procedure, it is important to keep in mind that the interval between engraftment and studying the experiment will vary depending on the mast cell injection site. It's very important to respect such intervals in order to allow sufficient time for the engrafted cells to mature and take up the final location vivo.

This approach has provided and continues to provide important information about what mass cell and their product can do or cannot do in vivo. Please always keep in mind that working with live animals require specific training, appropriate review and approval of experimental protocols and strict adherence to the highest standards of animal care, including taking care to reduce the animal's pain and stress when you perform these procedures.