For HSC quantification, bone marrow cells were isolated from tibiae and femurs with staining buffer (PBS supplemented with 2.5% FCS) and strained through 70-μm meshes. Red cells were lysed by resuspending the cells in 10 ml red cell lysis buffer (MACS Miltenyi Biotec) for 10 min at room temperature. After centrifugation, the cell pellet was resuspended in staining buffer and nucleated cells were counted with 3% acetic acid (StemCell Technologies) on a Vi-Cell XR cell viability counter (Beckman Coulter). Bone marrow cells (10 × 106 cells) were resuspended in 200 μl of staining buffer containing the following antibody solution: FITC-conjugated lineage cocktail with antibodies against CD4 (clone H129.19, BD Pharmingen), CD3e (clone 145-2C11, eBioscience), Ly-6G/Gr-1 (clone RB6-8C5, eBioscience), CD11b/Mac-1 (clone M1/70, BD Pharmingen), CD45R/B220 (clone RA3-6B2, BD Pharmingen), Fcε R1α (clone MAR-1, eBioscience), CD8a (clone 53-6.7, BD Pharmingen), CD11c (clone N418, eBioscience), TER-119 (clone Ter119, BD Pharmingen) and CD41 (FITC, clone MWReg30, BD Pharmigen); c-Kit (PerCP-Cy5.5, clone 2B8, eBioscience), Sca-1 (PE-Cy7, clone D7, eBioscience), CD150 (PE, clone TC15-12F12.2, BioLegend) and CD48 (biotin, clone HM48-1, BioLegend). The samples were incubated for
