TIMP-1 protein levels in cell culture supernatants of orbital fibroblasts were measured in cells seeded in six-well culture plates at a final concentration of 5 × 105 cells/well. After stabilization for 48 hours, cells were treated with IL-1β (Biosource, Camarillo, CA) or growth factors (fibroblast growth factor [bFGF]; Biosource), platelet-derived growth factor (PDGF)-BB (Biosource), and transforming growth factor (TGF)-β1 (R&D Systems, Minneapolis, MN). In some experiments, cells were cotreated with IL-1β and pirfenidone (Sigma-Aldrich, St. Louis, MO) or dexamethasone (Sigma-Aldrich), as described. After treatment for 48 hours, the concentration of TIMP-1 in supernatants was determined using a sandwich ELISA method. Briefly, 96-well plates were precoated overnight at room temperature with a capture antibody (R&D Systems). After three washes, plates were blocked with phosphate-buffered saline (PBS; pH 7.4) containing 5% (wt/vol) sucrose and 1% (wt/vol) bovine serum albumin, followed by incubation with detection antibody (R&D Systems). After washing, plates were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 20 minutes and then incubated with color reagents A (substrate, hydrogen peroxide) and B (tetramethylbenzidine; R&D Systems) for 30 minutes. Absorbance was measured spectrophotometrically at 540 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). The concentration of TIMP-1 in each sample was determined by reference to a standard curve generated using known amounts of TIMP-1 (R&D Systems).