Human ifnγ elisa ready set go

CD4⁺ T cells (2 × 10⁴ cells/well) were stimulated with plate-bound anti-CD3 mAb (5.0 μg/ml, UCHT1; R&D Systems) and anti-CD28 mAb (1.0 μg/ml, 37407; R&D Systems) in flat-bottomed 96-well plates for 3 days. Concentrations of IL-10, IFN-γ, and IL-17A in the supernatants were measured using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s protocol (OptEIA Human IL-10 ELISA Kit II; BD Biosciences; Human IFN-γ ELISA Ready-SET-Go! and Human IL-17A ELISA Ready-SET-Go!; eBioscience). Cells were cultured in Gibco RPMI 1640 medium (Invitrogen, Paisley, UK) supplemented with 10% FBS (Equitech-Bio, Kerrville, TX, USA), 100 μg/ml l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μM 2-mercaptoethanol (Life Technologies, Carlsbad, CA, USA).

Peripheral blood mononuclear cells from IBD patients were isolated and cultured in RPMI medium 1640 (Gibco) containing 10% FCS (Pan Biotech), and 1% penicillin/streptomycin. Cells were stimulated with anti-human CD3 (eBioscience, clone OKT3) and anti-human CD28 (BD Pharmingen, clone CD28.2) at a final concentration of 1 µg/mL in the presence of 40 µg/mL VDZ or IgG1 isotype control. After 24 and 48 h, supernatant was collected, and quantification of cytokine production was performed by ELISA for IFN-γ (human IFNγ ELISA Ready-SET-Go, eBiosciences, Cat. 88-7316), IL-4 (Human IL-4 ELISA Ready-SET-Go, eBiosciences, Cat. 88-7046), IL-17a (human IL-17A ELISA Ready-SET-Go, eBiosciences, Cat. 88-7176), and IL-10 (BD OptEIA Human IL-10 ELISA Kit II, BD Pharmingen, Cat. 550613). In separate experiments, PBMCs from IBD patients (UC: n = 7; CD: n = 7) were cultured for 24 and 48 h and induction of cell death in the presence of 40 µg/mL VDZ or IgG1 isotype control was assessed using the cell death detection plus Kit (Roche Diagnostics, Mannheim, Germany).