CD45+CD33+CD19− AML blasts from 6 patients (2 patients from each risk group; supplemental Table 1) were stained with CD33-PE (clone AC104.3E3; Miltenyi Biotec), CD45-FITC (clone HI30; BD Biosciences), and CD19-APC (clone HIB19; BD Biosciences) antibodies and sorted using a FACSAria Fusion Cell Sorter (BD Biosciences). Sorted blasts were plated using a limiting dilution analysis of 4 doses (250, 500, 1000 and 2000 cells) in 15 replicates in 96-well microplates containing a confluent irradiated (50 Gy) MS-5 monolayer. The AML blast-MS5 coculture was maintained in MyeloCult H5100 culture medium (STEMCELL Technologies) supplemented with recombinant human interleukin-3, granulocyte colony-stimulating factor, and thrombopoietin (MS-51 3GT) (20 ng/mL each; PeproTech), as previously described41 (link) at 37°C in 5% CO2. After 5 weeks, the number of wells plated with 250 cells displaying cobblestone areas (CAs) were scored, and the long-term culture (LTC) medium was replaced by methylcellulose H4435 (STEMCELL Technologies). After an additional 2 weeks, each well was scored as positive/negative if colony-forming units were present/absent. The frequency of LICs was determined using ELDA software, as previously described (http://bioinf.wehi.edu.au/software/elda/).33,42,43 (link)