Dual luciferase reporter 1000 assay system

The blocking activity of anti-IFNα2 and anti-IFNω autoantibodies was determined with a reporter luciferase activity as described previously⁶⁴ . HEK293T cells were transfected with a plasmid containing the firefly luciferase gene under the control of the human ISRE promoter in the pGL4.45 backbone and a plasmid constitutively expressing Renilla luciferase for normalization (pRL-SV40). Cells were transfected in the presence of the X-tremeGENE9 transfection reagent (Sigma-Aldrich, 6365779001) for 24 h. Cells in Dulbecco’s modified Eagle medium (DMEM; Thermo Fisher Scientific) supplemented with 2% fetal calf serum and 10% healthy control or patient serum/plasma (after inactivation at 56 °C for 20 min) were stimulated with IFNα2 (Miltenyi Biotec, 130-108-984) and IFNω (Merck, SRP3061) at 10 ng ml⁻¹ or 100 pg ml⁻¹ for 16 h at 37 °C. Each sample was tested once for each cytokine and dose. Finally, cells were lysed for 20 min at room temperature, and luciferase levels were measured with the Dual-Luciferase Reporter 1000 Assay System (Promega, E1980) according to the manufacturer’s protocol. Luminescence intensity was measured with a VICTOR-X Multilabel Plate Reader (PerkinElmer Life Sciences). Firefly luciferase activity values were normalized against Renilla luciferase activity values.