Competitive reconstitution assay to assess hematopoietic stem cell homing. (A) Schematic representation of competitive repopulation experiment. BM nucleated cells (10 million) from CD45.1 congenic WT mice were treated with rat IgG, anti–α4 integrin, anti–PSGL-1, or both anti–α4 integrin and anti–PSGL-1, and injected into lethally irradiated (12 Gy, single dose) E^–/– mice (CD45.2). Femoral bone marrow cells were harvested 3 hours after injection. Bone marrow cells contained in one femur were injected together with 1 to 2 × 10⁵ CD45.2 WT competitor BM cells into a lethally irradiated (12 Gy, split dose) CD45.2 WT secondary recipient mouse. Blood was harvested monthly from secondary recipient mice, and the expression of CD45.1 and CD45.2 was assessed by flow cytometry to determine the repopulating unit (RU). (B) Representative FACS dot plots 3 months after secondary transplantation. The left upper quadrant of each panel shows CD45.1⁺ leukocytes which were derived from long-term repopulating stem cells homed in the BM of E^–/– primary recipient mice. Many CD45.1⁺ leukocytes derived from homed repopulating cells can be observed in control rat IgG and anti–PSGL-1–treated groups. However, in all mice from the groups treated with anti–α4 integrin or both anti–α4 integrin and anti–PSGL-1, very few leukocytes were derived from cells homed in BM of E^–/– first recipients. Similar results were found up to 6 months after secondary transplantation. (C) RU levels following secondary transplantation. RUs were drastically reduced when α4 integrin was inhibited in the absence of E-selectin. n = 4 to 7 mice at one month and n = 3 to 6 at 6 months after secondary transplantation. Data are pooled from 3 independent experiments. *P < .05 for anti–α4 integrin and anti–α4 integrin plus anti–PSGL-1 groups compared with either IgG control or anti–PSGL-1 groups. #P < .05 compared with IgG control.