Quantification of IFN-β, RANTES, and TNF-α in Cell Culture

IFN-β protein in cell culture supernatants was measured using a custom ELISA originally described elsewhere (68 (link)), with few modifications. In brief, 96-well polystyrene plates (Maxisorp; Nunc International) were coated overnight with a 1:4,000 dilution of rat anti–mouse IFN-β mAb (Yamasa) in 0.1 M sodium carbonate at 4°C. Plates were blocked with 10% FCS in 1× PBS for 2 h at room temperature. Samples and a mouse IFN-β standard (National Institutes of Health [NIH]) were added to wells and incubated overnight at 4°C. Plates were washed 3 times with 1% FCS/PBS-T, followed by incubation with a 1:2,000 dilution of rabbit anti–mouse IFN-β pAb (PBL Biomedical Laboratories) in 10% FCS-PBS overnight at 4°C. Wells were washed 3 times, followed by incubation with a 1:2,000 dilution of goat anti–rabbit horseradish peroxidase (HRP; Cell Signaling Technologies) in 10% FCS-PBS for 1 h at room temperature. Plates were washed 3 times and developed with TMB substrate (KPL). The reaction was stopped by addition of 1 N H₂SO₄, and plates were read at 450 nm. For quantification of RANTES and TNF-α, Luminex bead-based colorimetric assays were performed by the Cytokine Core Laboratory (University of Maryland, Baltimore).