Bone Marrow Chimeras Protocol
(with hints from Jon Sprent)

1. Choose recipient C57BL/6 mice that are 6-10 weeks old.
2. The night before irradiation, remove their food but not their water.
3. Next morning, put 3 mice per clean Tupperware container - 1 in each division. Wipe down each container with 70% EtOH, and then add some bedding to the containers so that the mice are comfortable.
4. Irradiate 1 container at a time at 900 Rads (ca. 2 minutes per container). 5. Take mice back to colony, but still don't give them any food yet.

DONOR CELLS: (Need 10 million/mouse)
1. Prepare bone marrow from donors. From 8 bones (femurs & tibias), I recovered ca. 180 million cells. Put bones in a dish of sterile HBSS. Flush marrow out of the bones with a syringe/27g needle into another dish of HBSS. Pass marrow through a 22g needle to break up the clumps. Spin down the cells and resuspend in media.
2. Kill mature T cells - either by anti-Thy1 + complement, or by removing CD4+ and CD8+ cells with Dynabeads. Estimate 5% T cells in the bone marrow for your calculations. I resuspended the bone marrow in 50% media/50% HBSS and incubated them with 5 beads/T cell for 1-2 hr. Then I magnetized the solution to remove the beads.
3. Filter the T-depleted solution through 70 mm mesh to remove clumps. Spin down cells and resuspend in RPMI with no additions to a concentration of 10 million cells per 150 ml. Store on ice until injection.

1. Inject 150 ml cells (10 million) i.v. through the tail vein. Heat the mice up first with the heat lamp until they exhibit whisker-grooming behavior. Then make sure that they can be shaded under the cage insert, such that they don't get too hot.
2. Give them their food back. MAINTAIN THEM ON ANTIOBIOTIC-WATER.
3. Analyze them after at least 6 weeks. Their fur should turn gray in spots if they were properly irradiated. If the donor cells don't take, they will die around 10 days after irradiation.

Dawne Page, Ph.D.

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