Mercaptoethanol

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.

The DT40 cell line was derived from chicken B lymphoma [30 (link)] and was cultured in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% heat-inactivated FBS (fetal bovine serum), 1% chicken-serum, 50 μM mercaptoethanol (Nacalai Tesque), L-glutamine (Nacalai Tesque), 50 U/ml penicillin, and 50 μg/ml streptomycin (Nacalai Tesque). The cell line was maintained at 39.5°C in a humidified atmosphere and 5% CO₂. TK6 cell line is a human B lymphoblastoid line [31 (link)] and cultured in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 5% heat-inactivated horse-serum, L-glutamine (Nacalai Tesque), 0.2 mg/ml Sodium pyruvate (Sigma-Aldrich), 50 U/ml penicillin, and 50 μg/ml streptomycin (Nacalai Tesque). The TK6 cells were maintained at 37°C in a humidified atmosphere and 5% CO₂. The list of the mutant clones we analyzed in this manuscript is shown in Table 1.