Qualitative and quantitative assessment of HSCs from different homeostatic BM compartments. (A) Percent of HSPC subpopulations from different BM compartments, ± SD, quantified by flow cytometry from wild-type animals (n = 14; C57Bl6 or β-actin-luciferase mice). (B) Noninvasive BLI tracking of 1250 LSK cells sorted from Dia, Epi, or Cal, 7 days postintravenous transplantation in sublethally irradiated NOD/SCID recipients. Signal localization is observed in BM compartments, such as the femur, tibia, vertebrae, and calvaria. (C) Noninvasive BLI quantification of hematopoietic reconstitution. Animals injected with 1250 LSK cells from the designated source (Dia, Epi, or Cal) were imaged after 1, 2, and 6 weeks postinjection. Data displayed are the average of BLI activity (photons per second) from 4 animals at week 1, 3 animals at week 2, and 2 animals at week 6, ± SD. No statistical difference was observed between these 3 groups. Negative controls included noninjected animals (Non Inj), sublethally irradiated NOD/SCID injected with lineage low Sca-1–negative sorted cells (Sca neg), or 1250 LSK from diaphysis injected in nonirradiated NOD/SCID recipients (Non irrad). (D) Chimerism of lethally irradiated CD45.2 primary recipients 13 weeks after being transplanted with 0.5 × 10⁶ BM-leukocytes derived from the legs (L) or calvaria (C) of congenic (CD45.1) donors. Each circle represents the donor-derived chimerism level from each recipient (n = 5-6 per group) within the total leukocytes (CD45), B cells (B220), T cells (CD3), and myeloid cells (CD11b + CD11c). (E) Competitive secondary transplantation: percentage of chimerism of lethally irradiated CD45.2 recipients, 19 weeks after being transplanted with 10 × 10⁶ unfractionated BM cells from the legs of primary recipients from (D) (n = 4-5 per group).