Increased ROS levels in long-lived human HSCs. (A) Lin⁻CD34⁺CD38⁻ cells were isolated from CB (n = 4) and recipient mouse bone marrow at 18 weeks after primary (n = 4) and secondary transplantation (n = 4). Intracellular ROS concentrations were determined by the intensity of DCF-DA staining using a flow cytometor. The relative ROS level is presented as a fold induction compared with the MFI value detected in Lin⁻CD34⁺CD38⁻ cells isolated from CB (*P < .05). (B) Representative sorting profiles of DCF-DA stained Lin⁻CD34⁺CD38⁻ cells isolated from secondary recipients' bone marrow cells. Gray shade indicates a DCF-DA–staining of Lin⁻CD34⁺CD38⁻ cells isolated from CB. Purified DCF-DA^high cells and DCF-DA^low cells were immunostained for γ-H2AX. The box and whisker plot shows the number of γ-H2AX foci per cell. More than 50 cells in random fields on a slide were counted for 3 independent experiments (**P < .01). Bars represent 5 μm. DCF-DA^low and DCF-DA^high Lin⁻CD34⁺CD38⁻ cells isolated from secondary recipient were transplanted into sublethally irradiated NOG mice. Twelve weeks after transplantation, engraftment levels of human hematopoietic cells were assessed by flow cytometry (3 recipients in each group; **P < .01). (C) Apoptosis in Lin⁻CD34⁺CD38⁻ cells was assessed by annexin V/PI staining (n = 3 in each group). Inserted images demonstrate the immunostaining results of Lin⁻CD34⁺CD38⁻ cells from secondary recipient. Positive staining for γ-H2AX (green) but not for active form caspase-3 (red) was detected.