Mouse granulocyte macrophage colony stimulating factor mgm csf

Bone marrow-derived DCs (BMDCs) were generated by cultivating bone marrow cells in RPMI 1640 (Thermo Scientific, Waltham, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Scientific, Waltham, USA), 1% Penicillin/Streptomycin solution (Sigma-Aldrich, City of Saint Louis, USA), 20 ng/mL mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) (R&D, Minneapolis, USA) and 10 ng/mL mouse interleukin-4 (mIL-4) (R&D, Minneapolis, USA) for 7 days. The cells were stimulated with 10 μg/mL MO, 10 μg/mL Omp19, 0.1 μg/mL LPS (Sigma-Aldrich, City of Saint Louis, USA) or PBS for 24 h, respectively. The expression levels of CD11c (FITC Hamster anti-mouse CD11c, BD Pharmingen, clone HL3, Franklin Lakes, USA), MHC II (Alexa Fluor^® 647 Rat anti-mouse I-A/I-E, BD Pharmingen, clone M5/114.15.2, Franklin Lakes, USA), and CD80 (PE Hamster anti-mouse CD80, BD Pharmingen, clone 16-10A1, Franklin Lakes, USA) on DCs were evaluated by BD canto II (BD Biosciences, USA). The data were analyzed in FACS Diva™ Software.

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.