Mouse scf

Human AML cell lines were procured from ATCC or DSMZ and authenticated by ATCC using short tandem repeat DNA analysis. MOLM-13-Luc cell line expressing luciferase was established at Amgen Inc. (31 (link)). MOLM-13-Luc cells were negative for mycoplasma by DNA analysis (Charles River), the remaining AML cell lines have not been recently retested for the presence of mycoplasma. Cell lines were grown using recommended culture conditions and maintained at 37°C with 5% CO₂, a subset of lines were grown in the presence of penicillin/streptomycin (Invitrogen). Primary human bone marrow mononuclear cells (LONZA) were grown in 15% FBS in IMDM media supplemented with 2.5 ng/mL G-CSF, 2.5 ng/mL GM-CSF, and 10 ng/mL SCF (R&D Systems). To differentiate primary mouse bone marrow cell cultures to megakaryocytic lineage, c-kit+ progenitor cells were positively enriched from whole bone marrow obtained from Jak2^V617F knock-in mice using MACS system (Miltenyi Biotech). Enriched c-kit+ progenitor cells were grown in StemSpan Media (Stemcell Technologies) supplemented with 10 ng/mL mouse IL-3, 10 ng/mL human IL-6, 40 ng/mL mouse SCF, and 20 μg/mL human low-density lipoprotein (Stemcell Technologies) for 24 hours. Megakaryocytic differentiation was performed by culturing cells in 10% FBS in RPMI1640 media supplemented with 50 ng/mL mouse thrombopoietin (Stemcell Technologies).