Irradiated Mouse Model for Hematopoietic Stem Cell Transplant

11 host WT mice (male) ranging from 13 to 17 weeks of age were irradiated twice with 6.02 Gy in 4 h (Faxitron CP-160). 3 WT (male) and 3 Cxcl4^−/− (male) graft mice were sacrificed after isoflurane narcosis. Femora and tibiae were separated from muscle tissue and cleaned. Under sterile conditions, bone marrow was flushed out with syringes using phosphate buffered saline (PBS) with 2% fetal calf serum (FCS). Erythrocytes were lysed by incubating 5 min in 1x erythrocyte lysis buffer (BD Pharm Lyse) followed by two washing steps with PBS. Cell suspension was filtered through a 40 μm cell strainer. Tyrosine kinase KIT positive (cKIT+) cells were isolated by magnetic cell separation using murine cKIT-microbeads (Miltenyi Biotech) according to the manufacturer’s instructions. WT or Cxcl4^−/− cKIT⁺ stem cells were transplanted into the lethally irradiated host mice 2 h after the second radiation by retroorbital injection of 5 × 10⁵ cKit⁺ cells per mouse (6x WT, 5x Cxcl4^−/−). To protect against infections during engraftment, antibiotics (Sulfadimethoxin & Trimethoprim, 95 mg/kg BW) were added to drinking water for 21 days after transplantation. 28 days after transplantation, success of hematopoietic stem cell engraftment was checked by taking blood samples and monitoring blood count. Mice were subjected to IRI or sham surgery as described before.