Single CD34-LSK cells were sorted into 96-well microtiter plates containing the SF-O3 medium supplemented with 10% FBS and multiple cytokines (10 ng/ml SCF, 10 ng/ml TPO, 10 ng/ml IL-3, and 3 u/ml EPO) and allowed to form colonies.

The effects of SNS-032, perifosine, or combination on the leukemia colony formation (CFU-L) in methylcellulose medium were examined using leukemic colony assay as previously described [3) in 600 Mu L of methylcellulose solution were incubated in the presence of the agents or an equivalent amount of medium at 37°C in a humidified atmosphere with 5% CO2. Primary leukemic cells were cultured in methylcellulose medium containing recombinant human (rh) stem cell factor (SCF) , granulocyte macrophage-colony-stimulating factor (GM-CSF) , and interleukin 3 (IL-3) at 2 × 104 cells/dish. After 7 days, CFU-Ls that contain >40 cells were scored manually under a light microscope. For colony assay of human normal bone marrow cells, 3 U/mL rh erythropoietin, 50 ng/mL rhSCF, 30 ng/mL rhGM-CSF, and 10 ng/mL rhIL-3 were added to the methylcellulose medium. The colonies were counted under a microscope on day 12 of culture.

celltype NA >Leukemic cell lines, MNCs from the patients with, or healthy controls were infected with or without the indicated viruses (50 MOI) and seeded in triplicate in a mixture containing 1. 35% methylcellulose in medium NA >Iscove NA s medium NA >modified medium NA >Dulbecco NA s medium (IMDM;) supplemented with 20% fetal bovine serum and10−4 M 2-Mercaptoethanol. For colony assay of bone marrow cells, 3 U/mL recombinant human (rh) erythropoietin, 50 ng/mL rh stem cell factor, 30 ng/mL rh granulocyte macrophage–colony-stimulating factor, and 10 ng/mL rh interleukin-3 were added to the methylcellulose medium. The colonies were evaluated under a microscope on day 12 of culture. In contrast, leukemic cell lines were seeded in cytokine-free mixture as described previously [