The overall goal of this procedure is to rapidly induce type one diabetes in mice by transferring diabetogenic T cells from transgenic mice into mice, which lack endogenous t and b cells. Begin by harvesting the spleens and lymph nodes from the donor mice and purifying the CD four positive T cells. Next adoptively, transfer the purified donor T cells into non skid recipient mice.
Then monitor the recipient mice for type one diabetes development. Ultimately, this protocol can be used to study diabetes pathogenesis and to investigate therapeutic interventions Compared to other methods. The main advantage of this technique of transferring BDC 2.5, CD four positive T-cells into non-skid recipients is the rapid and reproducible development of type one diabetes.
For donors of the Diabetogenic CD four positive T cells use six week old pre-diabetic female BDC 2.5 mice that have been determined to be diabetes free by urine glucose measurement. To remove the spleen and lymph nodes, soak the fur with 70%ethanol, then cut the skin, retract the skin until the muscles of the front limbs are exposed. Near the base of the front limbs, two lymph nodes on each side should be visible Carefully Grasp each lymph node with small tweezers and remove it by trimming the connective tissue.
Next, make a one inch incision in the peritoneum using the small scissors. Now gently grasp the spleen in the center. Carefully trim away the connective tissue and the attached fat.
Then remove the spleen, lace, the spleens and lymph nodes in 10 milliliters of DMEM on ice. With the end of a sterile 10 milliliter syringe plunger, gently press the lymphoid organs through a 70 micrometer cell strainer. To obtain a single cell suspension.
Rinse each strainer with one milliliter of DMEM several times to maximize the recovery of cells. Then transfer the collected cells into a 15 milliliter conical tube and centrifuge at 400 Gs for seven minutes. At room temperature, re suspend the cell pellet in three milliliters of a CK buffer.
Now incubate every room temperature for five minutes. To lye the red blood cells now add 12 milliliters of DMEM and pellet the cells by centrifugation. Wash the cell pellet once in 10 milliliters of DMEM.
Next, we suspend the cells in five milliliters of fact staining buffer, and use a phase contrast microscope and hemo cytometer to count the number of viable cells. Now remove about one times 10 of the six cells per staining control. Also stain the cells with mouse monoclonal antibodies to sort for nonactivated transgenic CD four positive T-cell.
Incubate all those samples at four degrees Celsius in the dark for 30 minutes. Now wash the cells with three times the staining volume of fax buffer and resuspend the cells in fax buffer one to two times 10 to the seven cells per milliliter for sorting sample and 300 microliters for single stain controls. Next, pass the cell sample through a 35 micrometer cell strainer cap tube.
To remove cell clumps process the samples for a cell sorter with a trained operator Allow about three hours, including setup time to sort about 1.5 times 10 to the eighth totals Transgenic naive CD four positive T cells. You can expect about 2.5 times 10 of the six cells per mouse and the sort rate's gonna be about two times 10 of the fourth events per second on an instrument such as the BD Fox Aria. Collect the sorted cells into three milliliters of DMEM.
Record the absolute number of sorted cells and centrifuge them for seven minutes. At 400 Gs, we suspend the cell pellets in sterile phosphate buffered saline for the adoptive T-cell transfer. Using a one milliliter syringe in an 18 gauge needle, gently resuspend the fax purified CD four positive T cells.
Load the one milliliter syringe then in preparation for injection, attach a 27 point a half gauge needle to the syringe. Restrain the knot skid recipient mice and wipe their tail with 70%ethanol to disinfect the injection site. Then inject one times 10th of the six purified CD four positive T-cells per mouse into either of the lateral tail veins.
When doing your tail vein injections, make sure that you don't force the plunger. If the needle is actually in the vein, your injection should occur with almost no resistance Beginning five days after the T-cell transfer. Monitor the nod skid recipient mice daily for development of type one diabetes.
Measure the urine glucose using reagent strips according to the manufacturers and instructions. Note that mice with two consecutive urine glucose readings of greater than 250 milligrams per deciliter are considered diabetic. In a typical experiment, each BDC 2.5 donor mouse yields about two and a half times 10 of the six naive transgenic CD four positive T cells after cell sorting after adoptive transfer of diabetogenic CD four positive T cells, the mice were monitored for urine glucose concentrations.
The adoptive transfer of small numbers of cells from BDC 2.5 donor mice induced type one diabetes in all non-skid recipients by day 11. When done properly, this technique can be done in approximately eight hours from organ harvest to T-cell transfer.
