Inflammatory bowel disease, or IBD, imposes a considerable health and financial burden on individuals and the society. This colitis model is helpful to study the mechanisms of IBD and evaluate the treatments for IBD. This immunodominant Cbir flagellin T-cell-specific adoptive transfer colitis model provides insights into how gut bacteria antigen induces T-cell responses to induce colitis.
After euthanizing the mouse, make an approximately one-centimeter left abdominal incision and pull the skin away from the abdominal muscle tissue. Then, make an approximately three-centimeter incision in the abdominal muscle tissue. Next, remove the spleen with sterile scissors and forceps.
Place the spleen in a culture dish containing five milliliters of pre-cooled washing buffer. Grind the spleen with the rough surface of two sterile glass slides. Transfer the cell suspension into a 50-milliliter centrifuge tube by passing it through a 100-micrometer cell strainer.
Rinse the glass slides and culture dish with five milliliters of pre-cooled washing buffer, and transfer the washing buffer into the tube. Centrifuge the cell suspension, discard the supernatant, and resuspend the cells with five milliliters of prewarmed Tris ammonium chloride lysis buffer per spleen. Incubate for 10 minutes at room temperature.
Add 10 milliliters of pre-cooled washing buffer to the tube. Then, centrifuge the cell suspension, discard the supernatant, and resuspend the cells with 10 milliliters of pre-cooled isolation buffer. To count the cells, mix 10 microliters each of cell suspension and Trypan blue thoroughly, and load 10 microliters onto a slide.
Then, insert the slide into the automated cell counter to obtain the viable cell number. Centrifuge the remaining cell suspension and discard all supernatant. Vortex the anti-mouse CD4 magnetic particles, directly add 50 microliters of the particles per 10 million cells, and mix with cell pellets thoroughly.
Incubate the mixture for 30 minutes at four degrees Celsius. Transfer the cell particle suspension into a sterile collection tube. Add 3.5 milliliters of pre-cooled isolation buffer into the tube.
Place the tube on the cell separation magnet for eight minutes at room temperature. Then, carefully aspirate off the supernatant with a three-milliliter transfer pipette. Remove the tube from the cell separation magnet.
Then, resuspend the cells with 3.5 milliliters of pre-cooled isolation buffer and place the tube on the magnet for four minutes at room temperature. Carefully aspirate the supernatant with a three-milliliter pipette. Finally, resuspend the cells in one milliliter of pre-cooled FACS buffer.
To transfer the cells into the recipient mice, resuspend the Cbir1 TCR transgenic-naive CD4-positive T-cells to 5 million cells per milliliter in 1x PBS. Warm the mice under a heat lamp and restrain the mice using a mouse restrainer. Intravenously inject 200 microliters of the cell suspension into the tail vein of the mice.
After euthanizing the mouse, make an approximately one-centimeter ventral midline skin incision. Pull the skin away from the abdominal muscle tissue. Then, make a three-centimeter incision in the abdominal muscle tissue.
Next, identify the coecum and remove the entire colon with sterile scissors and forceps. Wet the colon with pre-cooled PBS in a culture dish. Incise the colon lengthwise, and rinse it with pre-cooled PBS.
Cut 1/3 of the colon longitudinally. Place the colon strip in a paper towel with the lumenal side facing upward. Perform Swiss-rolling using a toothpick.
Place the colon Swiss into a cassette, and put the cassette in 10%buffered formalin for 24 hours. Dehydrate and paraffin-embed the colon with an automated processor. Then, cut five-micrometer tissue sections on a microtome.
Mount the tissue on slides and perform hematoxylin and eosin staining. The Rag knockout mice began to lose weight around three weeks post-cell transfer, and their weight reached around 80 to 85%of their original weight six weeks post-cell transfer. Recipient mice showed short colon length six weeks post-cell transfer.
The recipient mice demonstrated more cell infiltration in the intestinal lamina propria four weeks post-cell transfer. They showed goblet cell loss and intestinal epithelial cell hyperplasia five weeks post-cell transfer, as well as mucosal erosion and inflammatory cell infiltration in the submucosa of the colon six weeks post-cell transfer. The histopathological scores increased substantially from four to six weeks.
Simultaneously, there was no inflammation in Rag knockout mice receiving PBS alone. Using this procedure, colonic cytokine levels could be determined by ELISA and qPCR.
