Murine interleukin 3 il 3

Ba/F3 cells and NIH/3T3 cells were obtained from the German Resource Centre for Biological Material (DSMZ). Phoenix E helper-virus free ecotropic packaging cells were a kind gift from G. Nolan, Stanford, USA. OP9 and M2-10B4 cells were a kind gift from Christine Dierks, University Hospital Freiburg, Germany. EL08-1D2 cells were kindly provided by R. A. Oostendorp, Munich, Germany. Primary murine stromal cells were isolated from C57BL/6 mice. Primary human stromal cells from healthy donors have been obtained from the stem cell transplant department of the University of Freiburg, Germany.
Ba/F3 cells were maintained in RPMI 1640 medium (Thermo Fisher, Waltham, MA, USA) containing 10% fetal calf serum (FCS; Sigma-Aldrich, St. Louis, MO, USA), 1% Penicillin-Streptomycin (PS; Thermo Fisher), and 2 ng/mL murine interleukin-3 (IL-3; Peprotech, Cranbury, NJ, USA). Phoenix E and NIH/3T3 cells were maintained in DMEM (Thermo Fisher) supplemented with 10% FCS. NIH/3T3 cells were cultivated in presence of 1% PS. OP9 cells and M2-10B4 cells were maintained in alpha-MEM (Thermo Fisher) supplemented with 10% FCS and 1% PS. EL08-1D2 cells were maintained in alpha-MEM supplemented with 7.5% FCS, 2.5% horse serum (Thermo Fisher), 1% PS und 200 µL ß-Mercaptoethanol (50 mM; Thermo Fisher). All cells were cultured at 37 °C with 95% O₂, 5% CO₂.

CSCcmBT549 cells were maintained, as described above. Differentiation was performed on a gelatin-coated dishes in DMEM containing 10% FBS, 0.1 mM MEM NEAA, 2 mM L-glutamine, 50 U/ml penicillin and streptomycin, 0.1 mM 2-mercaptoethanol, 10% conditioned media, 30 ng/mL murine stem cell factor (SCF) (PeproTech, Rocky Hill, NJ, USA), and 10 ng/mL murine interleukin-3 (IL3) (PeproTech, Rocky Hill, NJ, USA). The cells were passaged when it reached approximately 80% confluence. After 12 days, cells were analyzed for macrophage markers.