Antioxidant treatment prevents quantitative and qualitative loss of stem cell function in vitro and in vivo. (A) CB Lin⁻CD34⁺CD38⁻ cells were cultured with BSO or BSO plus NAC and immunostained for γ-H2AX (γ-H2AX: green; DAPI: blue). The number of γ-H2AX foci per cell was obtained. The pooled data from 3 independent experiments are presented as the box and whisker plot (**P < .01 relative). (B) CB Lin⁻CD34⁺CD38⁻ cells were cultured with BSO or BSO plus NAC for 2 days. The frequency of apoptotic cells was determined by annexin V/PI staining (n = 3 in each BSO concentration; **P < .01). (C) Cultured cells were pulsed with BrdU, and the BrdU incorporation was analyzed by flow cytometry (n = 4 in each culture condition; **P < .01). (D) Cells cultured under the indicated conditions were transplanted into NOG mice. Eight weeks after transplantation, engraftment levels of human hematopoietic cells were assessed by flow cytometry (6 recipients in each group; **P < .01). (E) Lin⁻CD34⁺CD38⁻ cells were isolated from secondary recipients that had been fed with control or NAC-containing food, and intracellular ROS concentrations were determined by the intensity of DCF-DA staining using a flow cytometer. The relative ROS level is presented as a fold induction compared with the MFI value detected in Lin⁻CD34⁺CD38⁻ cells isolated from control mice. Data were collected from 3 independent experiments (a total of 5 recipients in each group; *P < .05). (F) Lin⁻CD34⁺CD38⁻ cells were isolated from secondary recipients that had been fed with control or NAC-containing food, and immunostained for γ-H2AX. The number of γ-H2AX foci per cell was counted. The pooled data from 3 independent experiments are presented as the box and whisker plot (9 recipients in each group; **P < .01). (G) Lin⁻CD34⁺CD38⁻ cells isolated from NAC-treated or nontreated secondary recipients were cultured with cytokines for 3 days and then pulsed with BrdU. The percentage of BrdU positive cells was determined by flow cytometry. Data were collected from 3 independent experiments (a total of 9 recipients in each group; **P < .01). (H) The absolute number of Lin⁻CD34⁺CD38⁻ cells in the recipient mice treated with or without NAC (5 recipients in each group; **P < .01). (I) Primary recipients were injected with 1 × 10⁴ Lin⁻CD34⁺CD38⁻ cells isolated from CB. Secondary and tertiary transplantation was performed at the 18th week of engraftment. A total of 1 × 10⁵ CD34⁺ cells pooled from 2-5 reconstituted recipients was transplanted into new groups of irradiated recipients. Engraftment levels of human hematopoietic cells were assessed by flow cytometry. Black circles indicate recipients that had been treated with NAC. White circles indicate nontreated recipients. Data were collected from 4 independent experiments (a total of 8 recipients in each group; **P < .01).