Busulfan chimeras

6-12 week old mice were treated with 10mg/kg Busilvex (Pierre Fabre) intraperitoneally for 2 consecutive days with a 24 hour interval. 24 hours after the final dose, 1-2 x 10⁶ donor bone marrow cells were delivered intravenously into the tail vein in 100μl PBS. Donor bone marrow was processed by collecting and cleaning the femur and tibia of both legs from donor mice, and crushing in RPMI-1640 (BioWhittaker) using a mortar and pestle. Cells were washed through a 70μm strainer, a short red blood cell lysis with 0.83% ammonium chloride. B-cells, T-cells, NK-cells and neutrophils were removed by incubating cells at a concentration of 1 x 10⁷ cells in 70μl of MACS buffer and FC-block prior to incubation with biotinylated α-CD3, α-CD19, α-NK1.1, α-Ly6G (eBioscience). Cells were subsequently incubated with Streptavadin microbeads (Miltenyi Biotech) and separated using an LS-column (Miltenyi Biotech) on the magnetic field of a MACS Separator (Miltenyi Biotech). The negative fraction was collected, counted, and injected. Mice were reconstituted for a minimum of 40 days.