The overall goal of this procedure is to isolate lymphocytes from the mouse reproductive tract. This is accomplished by first removing the entire genital tract and mincing the tissue into small pieces to free the lymphocytes from the tissue. The finely minced tissue is digested with collagenase eight and vigorous stirring of the tissue at 37 degrees Celsius.
With a magnetic stir, the lymphocytes are then isolated by per call gradient. Ultimately, the lymphocytes can be characterized by flow cytometry Implication of this technique extended towards the diagnosis and the therapy of reproductive disease. As this method allow us to understand the behavior of tissue lymphocyte, which often are differ from the lymphocyte that isolated from circulated system.
Although this method provide insight into reproductive mucosa system, it can also apply to other systems such as intestinal and respiratory mucosa systems. After euthanizing a female mouse, use surgical scissors to make a low midline incision and then open the skin. Gently move the intestines aside, identify the ucs, and then isolate and remove the whole genital tract from the ucs to the vaginal open.
Place the genital tract into a Petri dish. Use a pair of scissors to open the entire tract to longitudinally. Then transfer the tissue to a Petri dish with complete RPMI 10 media.
Now cut the genital tract into fine pieces, and then transfer the pieces to a flask containing complete RPMI 10 and EDTA. Place the flask on a magnetic stir and stir the tissue two times for 15 minutes at room temperature each time to get rid of the epithelial cells after the last stir period, discard the supernatant and then pour the tissue pieces through a 40 micrometer cell strainer into a 50 milliliter conical tube. Wash off the EDTA residue with complete media.
Now transfer the filtered tissue pieces to a new flask with fresh RPMI 10 and collagenase and digest the pieces at 37 degrees Celsius on the magnetic stir set to vigorously stir the tissue after an hour, pour the supernatant through a 100 micrometer cell strainer into a fresh 50 milliliter conical tube and keep the filtered cell suspension on ice. Add fresh RPMI 10 with collagenase to the original flask and stir the tissue two more times using fresh complete RPMI 10 and collagenase each time. After the third digestion, no visible tissue pieces should be present in the solution.
Now, pool the supernatants from the three digestions together. Spin the cells for five minutes at 410 Gs and four degrees Celsius, and then discard the supernatant. Begin by resus suspending the cell pellet in a fresh 40%per call solution.
Add the cell suspension with 40%per call into a gradient tube, and then carefully underlay and equal volume of freshly prepared. 70%per call. Now centrifuge the gradient samples for 20 minutes at room temperature with the break off.
After centrifugation carefully harvest the lymphocytes at the interface layer between the perol gradients. Then wash the recovered cells with RPMI one to three for 10 minutes at room temperature. Finally, after discarding the sup natant re suspend the lymphocytes in the appropriate solution according to the desired experimental procedure.
This dot plot is an example of a lymphocyte population that was isolated from the genital tract of a chlamydia urederm intravaginally infected mouse. Seven days after infection, the single cell suspension was gated on CD three positive lymphocytes, and analyzed for CD four and CD eight expressing cells After its development. This technique paved the way for the researcher to explore reproductive lymphocyte function and the cell characteristics in virus mouse disease model that mimic human diseases.
