Human fibrinogen

Fibrin–FN matrices were prepared as described previously (Corbett and Schwarzbauer, 1999 (link); Midwood and Schwarzbauer, 2002 (link)) by mixing components to yield the following final concentrations: 600 μg/ml human fibrinogen (BioVision Inc, Milpitas, CA), 150 mM sodium chloride, 20 mM calcium chloride, and 10 mM Tris-HCl, pH 7.4. No additional FN or Factor XIII was required for clot formation. Cells were resuspended in a 0.025 M HEPES/0.13 M NaCl solution and added to the clot components for a final concentration of 1.2 × 10⁶ cells/ml. After thrombin addition, the mixture was quickly pipetted into BSA-coated wells and incubated at 37°C. After 30 min at 37°C, FN-depleted medium was added to the wells and clots were carefully detached from the well walls using a thin needle under a dissecting microscope. The diameter of the clot was measured over 4 h as described previously (Corbett and Schwarzbauer, 1999 (link); Midwood and Schwarzbauer, 2002 (link)). The percentage contraction for each condition was normalized to the contraction of a clot that did not contain cells. For treatment of cells with TGF-β1, fibroblasts were grown for 24 h in medium containing 10 ng/ml recombinant TGF-β1 protein or an equivalent volume of vehicle before use in contraction assay.