Generation and Purification of Flt3L-DCs

BMDCs were cultured as previously described with minor modification [59 (link)]. Bone marrow cells collected from femurs and tibias were cultured in RPMI-1640 medium (HyClone) supplemented with 10% FBS (Gibco), 50 μM 2-mercaptoethanol (Sigma–Aldrich), penicillin and streptomycin (Invitrogen) in the presence of 10 ng/ml mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF; R&D) and 10 ng/ml mIL-4 (R&D). The culture medium was replenished on Day 3, and the cells were harvested on Day 7. Bone marrow cells were cultured in the presence of 150 ng/ml mFlt3L (R&D) and harvested on Days 8−9 to collect Flt3L-DCs. For p38α deletion in Flt3L-DCs derived from p38α^CreER mice, 0.5 μM (Z)-4-hydroxytamoxifen (4-OHT; Sigma–Aldrich) was added on Day 4. The purity of both CD11c⁺ BMDC populations was >80%. Flt3L-cDC1s (CD11c⁺ B220⁻CD24⁺CD11b⁻) were sorted by FACS for coculture.