Generation of Dendritic Cells (DCs) from Mouse Bone Marrow

Generation of DCs from mouse bone marrow was performed according to Lutz et al. (21). At day 0, femurs and tibiae of 8- to 10-week-old female BALB/c mice were removed and purified from surrounding skin and muscle tissue. For disinfection, intact bones were left in 70% ethanol for 5 min and were washed with RPMI 1640 afterward. Subsequently, the articular heads of each bone were cut off and the bone marrow was flushed out. After cell clusters had been disintegrated, the cell suspension was centrifuged (350 g, 5 min, room temperature). Then, the cell pellet was resuspended in R10 supplemented with β-mercaptoethanol (0.05mM) and freshly added 200 U/ml mouse GM-CSF (referred to as DC medium). Cells were counted and 2 × 10⁶ bone marrow leukocytes were seeded per 100 mm PS bacteriological Petri dish (Falcon^®, Corning, NY, USA) containing 10 ml DC medium. At day 3, 10 ml fresh DC medium was added per plate. At days 6 and 8, half of the SN per plate was collected and centrifuged. Thereafter, the cell pellet was resuspended in 10 ml fresh DC medium and returned to the plate. At day 10, DCs were harvested.