The overall goal of this procedure is to induce intestinal inflammation by the adoptive transfer of naive T cells into immunodeficient mice. This is accomplished by first isolating cells from mouse spleens, lysing the blood cells and then enriching the population for CD four positive T cells. The second step is to label these cells with fluorescent DI conjugated CD four and CD 45 RB antibodies.
Next, the labeled cells are sorted using facts into CD 45, RB low and CD 45 RB high populations. The final step is to transfer the CD 45 RB high cells into immunodeficient mice by intraperitoneal injection. Ultimately, the transferred cells are activated and cause local tissue damage in the absence of T regulatory cells resulting in experimental colitis.
This method can help answer key questions in the study of inflammatory bowel diseases such as the role of specific cell populations and the gastrointestinal microbiota in disease pathogenesis. After euthanizing the donor mouse, spray the abdomen with 70%ethanol to sterilize the area. Then make a horizontal incision across the abdomen and peel back the skin to expose the peritoneum using forceps.
Hold the peritoneum away from the internal organs and make an incision in the left abdominal peritoneum. Next, excise the spleen from the mouse and place it in a Petri dish containing 10 milliliters of ice cold, complete medium. Crush the spleen with two sterile glass slides to make a single cell suspension.
Filter the cells through a 70 micron strainer into a 50 milliliter conical tube and then rinse the strainer with five milliliters of complete medium strain up to five spleens in one conical tube. Next, centrifuge the cells at 450 times G for seven minutes at four degrees Celsius. Aspirate the supernatant into a waste container and then gently resuspend the cells in 10 milliliters of ice cold labeling buffer.
Combine 20 microliters of the cell suspension with 180 microliters of 0.4%trian blue solution and incubate the cells for five minutes at room temperature. Then add 10 microliters of the cell suspension to a hemo cytometer and count the number of non blue cells under the microscope to begin the enrichment pellet and gently resus suspend the cells in ice cold labeling buffer at a concentration of 20 times 10 to the six cells per milliliter. Add five microliters of biotinylated CD four T cell enrichment antibody per one times 10 to the six cells, and then incubate the cells on ice with the antibody for 15 minutes.
Add 10 times the volume of labeling buffer to the cell suspension, and then centrifuge the cells at 450 times G for seven minutes. Carefully aspirate the supernatant without disturbing the cell pellet. Next, re suspend the streptavidin conjugated magnetic particles by vortexing and add five microliters of particles per one times 10 to the six cells to the cells.
Mix the particles with the cells by gently flicking the tube and then incubate the mixture at six to 12 degrees Celsius for 30 minutes. Resuspend the cells to a concentration of 20 to 80 times 10 to the six cells per milliliter with labeling buffer. Then transfer one milliliter of labeled cells to a 12 millimeter by 75 millimeter round bottom test tube and place this positive fraction tube on a magnet for six to eight minutes.
With the tube on the magnet, carefully remove the supernatant with a glass paster pipette without disturbing the labeled cells and transfer it to a new sterile 50 milliliter conical tube. This fraction contains the CD four positive T cells. Remove the positive fraction tube from the magnet and resuspend the cells in labeling buffer by pipetting vigorously.
Return the tube to the magnet for another six to eight minutes. Again, carefully transfer the SUP nain to the enriched fraction tube. Increase the yield of CD four positive T cells by repeating the enrichment as necessary to prepare the cells for labeling First centrifuge the enriched fraction at 450 times G for seven minutes and discard the supernatant.
Then resuspend the cells in labeling buffer to a concentration of 10 times 10 to the sixth cells per milliliter. Aliquot five times 10 of the fifth cells in individual micro fuge tubes for unstained isotope stained and single stained control cells. Then add five micrograms per milliliter of CD four, FE, and one microgram per milliliter of CD 45 RB PE antibodies to the tube containing the original cell suspension.
Add the isotope control stains and the single stains at the same concentration to the appropriate cell aliquots. Gently mix the cells with the antibodies and then incubate the tubes on ice for 30 minutes. Cover the tubes to protect them from light.
Next, wash the cells twice in labeling buffer as indicated in the text protocol and resuspend the cells at 10 times 10 of the six cells per milliliter in labeling buffer. Then pass the cell suspension through a 70 micron strainer into a fax tube. Place the tube on ice and cover it To prevent photo bleaching of the stains.
Use the unstained and single stage control cells to calibrate the compensation on the cell sorter. Exclude the non-viable cells using forward and side scattered gating. And then set the gating for CD four positive and CD four RB positive cells with the isotope stained controls.
Next gate, the CD four positive population and then sort the cells into CD 45, RB high and CD 45 RB low populations. Collect the cells in tubes containing two milliliters of complete medium and then run an aliquot of approximately 10 times 10 to the third cells from each fraction on the cell sorter to assess the sample purity to prepare CD 45 RB high and CD 45 RB low cells for injection wash and resuspend them in PBS to the appropriate cell density as indicated in the text protocol. Next, firmly restrain the recipient mouse and inject 100 microliters of the cell suspension intraperitoneal into the right and left sides of the abdomen.
Monitor the recipient mice for clinical parameters as indicated in the text protocol every week. Post injection disease progression is typically apparent at week five. Sacrifice a mouse at a predetermined time point or when it is lost 20%of its starting body weight and extract the colon.
Then measure and record the length and the weight of the colon. CD four positive CD 45 RB high T cells were transferred to rag one knockout and NFIL three RAG one double knockout recipient mice by five weeks post injection. Double knockout Mice had lost 10%of their initial body weight, colons of both rag one knockout and double knockout.
Recipient mice were thickened and shortened compared to colons from the control mice CD four positive CD 45 RB high T cells for a transfer to rag one knockout and rag one knockout P one 10 delta mutant recipient mice histological analysis at three weeks post transfer indicated epithelial hyperplasia, inflammatory cell infiltrates and crypt abscesses in rag one knockout delta mutant recipients but not rag one knockout mice Once mastered. This technique can be done in four to six hours if it is performed properly.
