2 mercaptoethanol me

Mediastinal lymph node (MLN) cells were cultured in Roswell Park Memorial Institute 1640 medium (Nacalai Tesque) supplemented with 10% fetal bovine serum (BioWest, Kansas City, KS, USA), antibiotic–antimycotic mixed stock solution (100 ×) from Nacalai Tesque, and 50 μM 2-Mercaptoethanol (ME) from Wako (Tokyo, Japan). MLN cells from HDM-sensitized mice were re-stimulated with HDM at 10 or 30 μg/mL. Five days later, IL-13, IL-5, IL-4, IL-2 and IL-17A in the culture supernatants were quantified by ELISA. Mouse conventional DCs (cDC) were induced from Bone-marrow cells of WT, Tlr4^−/−, Tlr9^−/−, Sting^−/− mice by the incubation with 100 ng/mL GM-CSF for 7 days.

KDR⁺CD34⁺ cells were sorted by FACS on day 6 of initial differentiation in a serum-and feeder-free culture system, as previously described^{26 (link),29 (link)}. In some experiments, 10 μM Ac-DEVD-CHO (Peptide Institute, Osaka) or 50 μM pifithrin-α (Wako) was added on day 0.
To induce HC and EC differentiation, the sorted KDR⁺CD34⁺ cells were then transferred onto fresh confluent OP9 cells^{26 (link),30 (link)}. For HC development, the sorted cells were cultured in α-MEM (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS) (Thermo Fisher Scientific), 50 μM 2-mercaptoethanol (ME) (Wako), 10 ng/ml thrombopoietin (TPO) (R&D Systems, Minneapolis MN, USA), 20 ng/ml interleukin-3 (R&D Systems), and 100 ng/ml stem cell factor (SCF) (R&D Systems) until day 20 of differentiation. Subsequently, 10 ng/ml granulocyte colony-stimulating factor (G-CSF, Kyowa Hakko Kirin, Tokyo, Japan) was added instead of SCF and TPO. Methylcellulose colony-forming assays were performed as previously reported^{29 (link)}. To analyze EC development, the sorted cells were cultured in α-MEM supplemented with 10% FCS, 50 μM 2-ME, and 20 ng/ml VEGF (R&D Systems).

The hiPSC line 201B7 was provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. The hiPSC lines, iPS-TIG120–3f7 iPS-TIG107–4f1 and iPS-TIG114–4f1, were provided by the Center for iPS Cell Research and Application, Kyoto University through the JCRB Cell Bank (National Institutes of Biomedical Innovation, Health and Nutrition, Japan). hiPSCs were grown on mitomycin C-treated mouse embryonic fibroblasts (MEFs; ReproCELL). MEFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Japan Bio Serum) on cell culture dishes coated with 0.1% gelatin (Sigma-Aldrich). hiPSCs were cultured with DMEM/F12 (Gibco) containing 20% KnockOut Serum Replacement (KSR; Invitrogen), 1% L-Glutamine (Invitrogen), 1 × penicillin/streptomycin (Sigma-Aldrich), 1 × MEM non-essential amino acids (MEM-NEAA; Sigma-Aldrich), 0.1  mM 2-mercaptoethanol (ME; Wako Pure Chemical Industries) and 5  ng/ml recombinant human fibroblast growth factor 2 (rhFGF2; Chemicon), with the medium changed every day. hiPSCs were passaged every week using a dissociation solution (CTK solution; ReproCELL), with a split ratio of 1:3.