E84-low LSK cells from bone marrow are enriched for HSCs. (A) Flow cytometry analysis of hematopoietic stem/progenitor cells (HSPCs) based on E84 staining. Bone marrow cells were stained using E84, LSK, and SLAM markers to detect LT-HSCs (LSK⁺CD150⁺CD48⁻), ST-HSCs (LSK⁺CD150⁺CD48⁺), and MPPs (LSK⁺CD150⁻CD48⁺). Left panel: gating of LSK cells into LT-HSCs, ST-HSCs, and MPPs; right panel: histogram of E84-stained LT-HSCs, ST-HSCs, and MPPs. (B) Gating of the 25% least and 25% most intensely E84-stained LSK cells for subsequent sorting. (C) Sorted cells were analyzed for SLAM markers (CD48 and CD150) and gated accordingly. The plot is the summary of 3 independent experiments. (D) Sorted cells were cultured in Iscove modified Dulbecco medium with 10% FBS supplemented with interleukin-3/interleukin-6/stem cell factor for cell viability assays. (E) Annexin V staining of sorted cells in culture D. (F) Sorted cells were used for colony formation assays; colony-forming units (CFUs) from 3 independent assays were counted and plotted. The data are shown as mean ± standard deviation. *P < .05; **P < .01.