Flt3 l

Purified and fresh CD34+ cells were incubated with the early cytokines TPO, SCF, and FLT3L (ProSpec, East Brunswick, NJ, USA) at a concentration of 50 ng/mL. After three days in expansion medium, cells were harvested and evaluated for cell proliferation.

Primary Ph⁺ ALL bone marrow cells were kindly provided by Dr Michael Caligiuri (Ohio State University, Columbus, OH) and Dr Martin Carroll (University of Pennsylvania, Philadelphia, PA). Cells were seeded on a substrate of adherent, mitomycin C-treated OP9 stromal cells and maintained in SFEM (Stem Cell Technology, Vancouver, Canada) supplemented with IL-3 (10 ng/ml), IL-7 (10 ng/ml), FLT3L (20 ng/ml) and SCF (30 ng/ml) (ProSpec, Israel).
CD34⁺ CML cells were kindly provided by Dr Tessa Holyoake (University of Glasgow, United Kingdom) and cultured in SFEM supplemented with IL-3 (20 ng/ml), IL-6 (20 ng/ml), SCF, and thrombopoietin (10 ng/ml).
Commercially purchased (Stem Cell Technologies) cord blood CD34⁺ cells were cultured in SFEM (Stem Cell Technologies) enriched with the CC100 cytokine cocktail (SCF, 100 ng/ml; FLT3L, 100 ng/ml; IL-3, 20 ng/ml; IL-6, 20 ng/ml).
Ph⁺ and normal CD34⁺ primary cells were kept in culture at 37 °C, under a 5% CO₂ humidified atmosphere. Cell counts were performed using 0.4% Trypan Blue Solution and a Neubauer hemocytometer.

Anti-phospho-Abl (pY245), anti-phospho-CRKL (pY207), anti-phospho-AKT (pS473), anti-total-AKT, anti-phospho-consensus peptide in AKT substrates (see Supporting Materials), anti-phospho-STAT3 (pY705), anti-STAT3, anti-phospho-p44/42-MAPK, anti-p44/42 MAPK, anti-cleaved caspase-3, anti-caspase 9, anti-PARP1, anti-cytochrome c, anti-MCL1, anti-BCL2, anti-XIAP and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Cell Signaling Technologies. Anti-BCLxL was from BD transduction Laboratory. Anti-COX4 was from GeneTex. Mouse anti-Abl monoclonal antibody (8E9) was generated in our laboratory. Anti-phospho-STAT5 and anti-phosphotyrosine (4G10) were from Upstate Cell Signaling. Anti-BIM was from Calbiochem. Imatinib was from Santa Cruz Biotech. Gefitinib was from LC Laboratories. Dasatinib and nilotinib were from Euroasia. MK2206 was from Selleck chemicals. SCF, IL3, IL6 and Flt3-L were from Prospec. EPO was from Calbiochem. Recombinant human basic fibroblast growth factor (bFGF) was from Peprotech. All the other reagents were from Sigma Aldrich.