Generation of BMDCs

Femur and tibiae from 12 to 16 weeks old mice were flushed with M2 medium and erythrocytes were lysed by incubation with 5 ml of diluted BD Pharm Lyse buffer^TM for 1 min at room temperature. 5 × 10⁶ bone marrow cells were plated in 10 ml M2 medium (containing 10% heat inactivated FCS, 50 μM 2-mercaptoethanol) and 10% GM-CSF (conditioned medium obtained as supernatant from B16 cells expressing GM-CSF; originally kindly provided by Georg Häcker, Freiburg, Germany) in 10 cm Petri-dishes. On day 3 and 6 cultures were replaced with 10 ml of fresh M2 medium containing 10% GM-CSF, respectively. BMDCs cultures were used for experiments on day 7.