Bone Marrow-Derived Dendritic Cell Generation

Bone marrow cells in the femur were pushed out into PBS using a 1 mL syringe with a 26G needle. The collected cells were cultured in an RPMI-1640 medium containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (Wako), 55 μM mercaptoethanol (Nacalai Tesque, Osaka, Japan), 10 mM HEPES (pH 7.2; Nacalai), and granulocyte–macrophage colony-stimulating factor (GM-CSF, 20 ng/mL; R&D systems, Minneapolis, MN, USA) for 6 days. An equal volume of complete medium containing 40 ng/mL GM-CSF was added to each well on day 3. After 6 days of culture, non-adherent cells were collected as the DC-enriched cell population. BMDCs were treated with LPS (InvivoGen, San Diego, CA, USA) or YB, followed by subsequent analysis. The YB was prepared from young barley leaf powder (Toyo Shinyaku) as previously described [50 (link)]. Cellulose and hemicellulose in the total fiber were 51.8% and 36.9% in the YB, respectively [9 (link)]. As a Dectin-1 antagonist, whole glucan particle (WGP) (InvivoGen) was used at the indicated concentrations.