Recombinant murine flt3l

To generate murine iCD103 DCs, bone marrow mononuclear cells after depletion of red cells were cultured in 10 mL volume of RPMI 1640 medium enriched with 10% heat-inactivated FBS (10099, Gibco), 40 ng/mL recombinant murine FLT3L (250-31 L, Peprotech) and 20 ng/mL recombinant murine GM-CSF (315-03, Peprotech) for 7 days, and subsequently replaced with the same combination of cytokines and collected at days 10 to 14 for subsequent experiments.

To generate Flt3-Ligand (FLT3L) dendritic cells, bone marrow was cultured for 8 days undisturbed in R10 culture medium supplemented with 55 μM 2-mercaptoethanol and 200 ng/ml recombinant murine FLT3L (PeproTech).

CD103⁺ DCs were induced as previously reported by Mayer et al.⁴⁴ Bone marrow cells were gathered from the tibia and femur of the mice. Cells were resuspended at 1 × 10⁶ bone marrow cells per 10 cm dish in complete RPMI‐1640 (Gibco) containing 10% fetal bovine serum (FBS, Excell), 1% antibiotic–antimycotic (Sigma), 50 μM β‐mercaptoethanol (Sigma), 20 ng/ml recombinant murine‐granulocyte–macrophage colony‐stimulating factor (GM‐CSF) (PeproTech), and 100 ng/ml recombinant murine Flt3L (PeproTech). On day 3, after the plate was rotated slightly, remove the nonadhesive cells and collect them by centrifugation, and replace the solution with 5 ml of fresh culture medium. The purity of the isolated cells was analyzed by flow cytometry using anti‐mouse CD11c antibody until harvest on the 15th to 16th day of culture.