Anti il 4 antibodies clone mab204

Peripheral blood mononuclear cells (PBMC) were isolated from blood donor derived buffy coats through gradient centrifugation (Lymphoprep, Axis shields diagnostics, Dundee, Scotland). Naive CD45RA + CD4⁺T-cells were subsequently isolated with magnetic bead separation using the “Naive CD4⁺T-cell Isolation Kit II, human” (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were then activated and polarized toward T_H1 using Dynabeads™. Human T-Activator CD3/CD28 (1 bead/cell) (Dynal AS, Lillestøm, Norway), 5 ng/μl recombinant human IL-12p70, 10 ng/μl recombinant human IL-2 and 5 μg/μl anti-IL-4 antibodies (clone MAB204) (all three from, Bio-Techne, Minneapolis, USA), in RPMI 1640 media (Gibco, Paisley, United Kingdom). A portion of T-cells used for RNA and protein isolation was obtained at baseline and after 0.5 h (only RNA), 1 h, 2 h, 6 h, 24 h and 5 days (only protein; point not used in the analysis). The cells were washed twice in PBS, snap frozen in a dry ice ethanol bath and stored at −80°C until use. During the protein and RNA extractions, multiple samples were pooled from twelve different individuals to reach the necessary amount of material for the subsequent analysis steps.

The pre-incubated cells were activated and differentiated towards T_H1 using Dynabeads™ Human T-Activator CD3/CD28 (1 bead/cell) (Dynal AS, Lillestøm, Norway), 5 µg/ml recombinant human IL-12p70, 10 µg/ml recombinant human IL-2 and 5 mg/ml anti-IL-4 antibodies (clone MAB204; all from, Bio-Techne, Minneapolis, USA). P4 was added together with the activation and differentiation mixture to the cells pre-incubated with P4 cells. The cells were incubated at 37°C, with 5% CO₂ in RPMI 1640 media containing L-glutamine, 10% FBS and 1% Penicillin/Streptomycin (all from Gibco^®, ThermoFisher Scientific). Cells were either cultured in 6-well (for RNA-seq) or 96-well plates (for ATAC-seq) in biological triplicates at a density of 1 million cells/ml. Cells were subsequently harvested at 0.5, 1, 2, 6 and 24 hrs and processed for RNA-seq and ATAC-seq. An overview of the study design is shown in Figure 1.