Immature DCs were prepared by culturing bone marrow precursors from C57BL/6 mice in RPMI 1640 supplemented with 20% FCS, 2 mM L-glutamine, 100 U/ml penicillin and streptomycin, 50 μM 2-mercaptoethanol, sodium pyruvate and nonessential amino acids in the presence of 100 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF). Medium was replaced every second day, cells were frozen at -140°C on day 7. Frozen DCs were thawed, counted and directly applied to T-cell stimulation assays.