Recombinant human il 12p70

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.