Differentiation of Murine Bone Marrow Cells

Bone marrow cells were collected from the femurs and tibiae of wild-type BALB/c mice (Taconic) by flushing the opened bones with Iscove's Modified Dulbecco's Medium (IMDM; Invitrogen). Red blood cells were lysed in dH₂O followed by the addition of 10X PBS. After centrifugation, the cells were washed once in PBS containing 0.1% BSA. The bone marrow cells were cultured at 10⁶/mL in media containing RPMI 1640 (Invitrogen) with 20% FBS (Cambrex), 100 IU/mL penicillin and 10 μg/mL streptomycin (Cellgro), 2 mM glutamine (Invitrogen), 25 mM HEPES and 1x non-essential amino acids and 1 mM sodium pyruvate (Gibco) and 50 μM β-mercaptoethanol (Sigma) and supplemented with 100 ng/mL stem-cell factor (SCF; PeproTech) and 100 ng/mL FLT3-Ligand (FLT3-L; PeproTech) from day 0 to day 4. On day 4, the media containing SCF and FLT3-L was replaced with media containing 10 ng/mL recombinant mouse interleukin-5 (rmIL-5; R&D Systems) only. On day 8, the cells were moved to new flasks and maintained in fresh media supplemented with rmIL-5. Every other day, from this point forward, one-half of the media was replaced with fresh media containing rmIL-5, and the concentration of the cells was adjusted each time to10⁶ /mL. Cells were enumerated at day 0 and on days indicated thereafter in a hemocytometer, and 50,000 cells were subjected to cytospin (Thermo Shandon, Pittsburgh, PA). The cytospin preparations were fixed and stained using a modified Giemsa preparation (Diff Quik, Dade Behring AG, Dudingen, Switzerland).