Recombinant human il 4

In order to obtain human monocytes, PBMCs were cultured in a 75 cm² cell culture flask for 2 h at 37 °C in a 5% CO₂ environment at a concentration of 3–4 × 10⁶ cells/mL in X-VIVO 15 media (Lonza, MD, USA) supplemented with 1% inactivated autologous serum, gentamicin (50 μg/mL, B/Braun Medical, Melsungen, Germany), fungizone (2.5 μg/mL, Life Technologies, California, USA), and Zidovudine (1μM, Genéricos Españoles Laboratorios, Madrid, Spain). Monocytes were confirmed to be in a monolayer after incubation for 2 h, and the nonadherent cells (MNC) were removed by three washes with phosphate-buffered saline (PBSx1) (Figure S1). Subsequently, monocytes were cultured for 6 days with 1000 U/mL each of recombinant human IL-4 (ProSpec, Rehovot, Israel) and recombinant human GM-CSF (ProSpec) on days 0 and 2. After 6 days, immature dendritic cells (iDCs) were obtained by adding cold PBSx1 to the 75 cm² cell culture flask to the harvest them by firmly tapping the flasks. This dislodged the loosely adherent dendritic cell (DC) clusters. MDDCs were harvested into a 50 mL conical tube and counted manually for viability using the trypan blue exclusion dye. Lymphocytes were collected from MNC fractions and frozen as PBMCs for use in mixed lymphocyte response assays across different DC preparations.

Minimal Essential Medium (MEM) and Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12) with glutamax were purchased from Gibco (Thermo Fisher Scientific, Breda, Noord-Brabant, The Netherlands). Fetal bovine serum (FBS) was purchased from Bodinco (Alkmaar, Noord-Holland, The Netherlands). The bovine collagen type I solution was obtained from Advanced BioMatrix (CellSystems Biotechnologie Vertrieb, Troisdorf, Germany). The sodium salts of propionate and butyrate, as well as fibronectin from human plasma and penicillin–streptomycin (pen-strep) solutions, were purchased from Sigma (Breda, Noord-Brabant, The Netherlands). Sodium acetate was obtained from Merck Millipore (Amsterdam-Zuidoost, Noord-Holland, The Netherlands). Recombinant human IL-13 were purchased from R&D Systems (Abingdon, Oxfordshire, UK). Recombinant human IL-4 was purchased from ProSpec-Tany TechnoGene (Ness-Ziona, Israel). The HDM extract was obtained from GREER (Lenoir, NC, USA). The following monoclonal antibodies: anti-JNK1 + JNK2 (phospho T183 + Y185) antibody, anti-ERK1/2 (phospho Thr202/Tyr204) antibody, anti-GAPDH antibody, rabbit anti-mouse IgG H&L (HRP) conjugated antibody and goat anti-rabbit IgG H&L (HRP) conjugated antibody were purchased from Cell Signalling Technology (Leiden, Zuid-Holland, The Netherlands) and Abcam (Cambridge, UK).