BM from FLT3^wt/ITDmice demonstrates enhanced myeloid colony-forming activity and can be immortalized in vitro. Methylcellulose-based in vitro colony-forming assay of total BM cells (A) and Lin^−/low BM cells (B) from 2-month-old mice; (C) Secondary and tertiary colony-forming assay; (D) Flow cytometic analysis of cells collected from colonies 11 days after the first plating and from the long-term liquid culture (6 months). (E) Cells derived from FLT3^wt/ITD BM could be serially propagated in RPMI 1640 medium containing SCF and IL-3. *P < .05. (F) BM cells from FLT3^wt/ITD mice have a lower cytokine requirement to form CFU-GM. Data are results from 3 independent experiments. Data are expressed as means (bars) plus or minus SEM (error bars). (G) Representative CFU-GM colonies generated from FLT3^wt/ITD or wild-type BM in the presence of 1 ng/mL GM-CSF. Original magnification: ×40. Images were acquired at room temperature using a Nikon Eclipse TE300 inverted microscope system with a Nikon Plan Fluor 4×/0.13 NA objective. The images were photographed using a Diagnostics Instruments Spot camera with SPOT 3.3.2 software (Diagnostic Instruments, Sterling Heights, MI).