One day after total body irradiation of the recipient BALB/c mice, place a CD45.1/Ly5.1 B6.SJL donor mouse in the prone position on a clean working sheath, and use one 13-centimeter curved and serrated tip of a Semken forceps to lift the fur at one Achilles heel.
Using hardened 8.5-centimeters fine scissors with straight tips, incise the skin between the forceps and one heel, elongating the incision cranially to facilitate removal of the skin and fur from the hind limbs. When the skin has been removed from the hind limbs, cut through each hip, ankle, and knee joint.
Store the thigh and shank of every hind limb in a single 92-millimeter Petri dish filled with PBS on ice, and carefully remove as much muscle as possible from each bone, transferring each cleaned femur and tibia into a 50-milliliter tube of sterile RPMI 1640 medium on wet ice.
To isolate the bone marrow, transfer one bone at a time into a new 92-millimeter Petri dish filled with fresh RPMI 1640 medium, and use a scalpel to cut the ends off of each bone. Next, use a 1-milliliter syringe equipped with a 26-gauge needle to flush the bones with one milliliter of fresh medium at a time into a 50-milliliter collection tube containing five milliliters of medium.
When all the bones had been flushed, gently pipette the crumbly bone marrow pieces up and down several times to generate a single-cell suspension. Before filtering the cells through a 40-micrometer mesh screen cell strainer into a new 50-milliliter collection tube, pellet the cells by centrifugation, and resuspend the cells in five milliliters of ammonium chloride potassium lysis buffer for a three-minute incubation at room temperature. At the end of the incubation, stop the reaction with 10 milliliters of PBS and centrifuge the cells again.
Resuspend the pellet in two milliliters of fresh PBS for counting, and set aside a 6 x 106 cell aliquot for downstream flow cytometric analysis. Next, use a commercially available cell purification kit to magnetically deplete the CD90.2-positive T cells from the bone marrow single-cell suspension according to the manufacturer's instructions, and set aside a 1 x 106 cell aliquot of the T cell-depleted eluate for downstream flow cytometric analysis of the cell purity and composition before and after separation.
Then, use a one-milliliter syringe equipped with a 30-gauge needle to inject 5 x 105 T cell-depleted bone marrow cells and 100 microliters of PBS intravenously into the retrobulbar space of the venous sinus of each irradiated recipient animal.
The next day, place a spleen harvested from a donor animal onto a 40-micrometer mesh strainer in a 50-milliliter collection tube, and use a syringe plunger to press the spleen tissue through the strainer into the tube. Wash the strainer and plunger with PBS to collect all the splenocytes, and pellet the cells by centrifugation, after red blood cell lysis as demonstrated. Resuspend the white blood cells for counting, and set aside a 6 x 106 cell aliquot for downstream flow cytometric analysis.
For a total CD3-positive T cell isolation from total splenocytes, use a commercially available cell purification kit according to the manufacturer's instructions, and set aside a 1 x 106 CD3-positive T cell aliquot from the positive cell fraction for downstream flow cytometric analysis of the cell purity and composition, before and after separation. Then, inject 7 x 105 alloreactive CD3-positive T cells and 100 microliters of PBS intravenously into the retrobulbar space of each recipient mouse to induce GvHD.
