3D Collagen Gel Culture of Orbital Fibroblasts

A 3D collagen gel culture environment was established by embedding cells in 1.5 mg/ml collagen type 1 matrix (First Link Ltd., Birmingham, UK), as previously described^{20 (link),21 (link)}, using 0.74 × 10⁵ cells/ml orbital fibroblasts alone or with macrophages at 1:2 (1.5 × 10⁵ cells/ml) or 1:5 (3.7 × 10⁵ cells/ml) ratios. For gel contraction assays and immunofluorescence experiments, 150 μl gels were cast in the microwells of glass bottom MatTek dishes (MatTek Corporation, MA, USA), and then detached as free-floating gels^{20 (link)}. For all other experiments, gels were cast in 24-well plates with 210 μl gel solution and left attached. Where appropriate, inhibitors (IGF-1R blocking antibody 1H7, 5 μg/ml, BioLegend, CA, USA; TGF-β inhibitor SB431542, 10 μM, Cayman Chemical, MI, USA; PI3K inhibitor LY294002, 10 μM, Cell Signaling Technology, MA, USA) or control diluent at matching concentration were added to the culture medium immediately after gel polymerization and maintained throughout the experiment (IGF-1R blocking antibody 1H7, 10 μg/ml was also added to the gel mix).