Isolation and Sorting of AML Stem Cells

8227 cells were stained with CD34–APC-Cy7 (BioLegend) and CD38–PE-Cy7 (BD Biosciences) and washed with PBS before sorting CD34⁺CD38⁻ and CD34⁻CD38⁺ cells. Cells were FACS-sorted on a MoFlo system (Beckman Coulter). Primary AML specimens were stained with BB515–CD45 (BD Biosciences, 564585) to identify the blast population, PE–CD19 (BD Biosciences, 555413) and PE-Cy7–CD3 (BD Biosciences, 557749) to exclude the lymphocyte populations, 4,6-diamidino-2-phenylindole (DAPI; EMD Millipore, 278298) as a dead-cell marker and CellROX deep red (Thermo Fisher Scientific, C10422), and sorted using a Sony SH-800 system. ROS-low LSCs were identified as the cells with the 20% lowest ROS levels and the ROS-high blasts were identified as the cells with the highest 20% ROS levels, as recently described in detail^{14 (link)}.