Recombinant human il 4 protein

THP‐1 cells were differentiated into M0 macrophages by incubation with 200 ng mL⁻¹ phorbol 12‐myristate 13‐acetate (Sigma) for 48 h. Then, the phorbol 12‐myristate 13‐acetate was removed and M0 macrophages were polarized into M2‐like macrophages by supplementing the growth media with 20 ng mL⁻¹ of recombinant human IL‐4 protein (R&D Systems Inc.) and 20 ng mL⁻¹ of recombinant human IL‐13 protein (R&D Systems Inc.) for 72 h. The cells were collected for RNA isolation and the supernatants from control and IL‐4 + IL‐13 macrophages were centrifuged at 1000g to remove cell debris and defined as M0(control) THP‐1 CM and M(IL‐4 + IL‐13)THP‐1 CM, respectively, which were used after addition of 30% fresh complete medium. Pooled human peripheral blood mononuclear cells were left to sit down on the plate THP‐1 media for 7 days and all adherent cells were washed with phosphate‐buffered saline and given either fresh THP‐1 media for M0(control)THP‐1 media with or M(IL‐4 and IL‐13) THP‐1CM to polarize toward M2 for 72 h. Cells were collected for flow cytometry analysis.

DCs were generated from the bone marrow cells of 8 weeks old female BALB/c mice. The bone marrow cells were centrifuged, washed and cultured in roswell park memorial institute 1640 medium (Hyclone, Utah, USA) containing 10% fetal bovine serum (Sciencell, CA, USA), 1% penicillin/streptomycin (Beyotime), 10 ng/ml recombinant human IL-4 protein (R&D systems, Minnesota, USA) and 20 ng/ml granulocyte macrophage-colony stimulating factor (PeproTech, NJ, USA) and incubated at 37 °C and 5% CO2 for seven days. Cells were cultured with recombinant human IL-17B protein (500 ng/mL) (R&D systems) for 24 h. Cells cultured with medium provided a negative control while cells cultured with LPS (10 ng/mL) (Sigma, Missouri, USA) provided the DC activation positive control. DC phenotypes were analyzed via surface expression of specific markers. Live DCs were immunolabeled with APC-CY7-livedead (Thermo Fisher Scientific, Massachusetts, USA), eFluor450-CD11C (Thermo Fisher Scientific), PE-CD80 (Thermo Fisher Scientific), APC-CD86 (Thermo Fisher Scientific), FITC-MHC II (Thermo Fisher Scientific) at 4 °C for 30 min. Resuspend cells were detected on Beckman Cytoflex LX and the result was analyzed by FlowJo 10.