Anti mcp 1

Neutralizing antibody anti-MCP-1, anti-M-CSF or both were added at day 0 and 3 at the concentration of 20 and 10 μg/ml, respectively (R&D Systems).

A chemoinvasion assay was performed to evaluate the ability of cells to cross a Matrigel membrane. The upper chambers, with 8 mm pores, were coated with 50 μl Matrigel diluted 1:10 (v:v) in DMEM and were incubated at 37°C for 4 h. The lower chambers contained either DMEM supplemented with 1% BSA as a control or UC-CM. For specific factor blocking assays, 20 μg/ml each of the monoclonal mouse anti-human anti-SDF-1 (cat. no. MAB350; R&D Systems, Inc.), anti-MCP-1 (cat. no. 16-7096; eBioscience, Inc.) and anti-HGF (eBioscience, Inc.) antibodies were added to the lower chambers. The fibroblasts, HUVECs and UC-MSCs were prepared in DMEM supplemented with 1% BSA, and 5×10⁴ cells in 0.5 ml suspension were added to each upper chamber. Each experiment was performed in triplicate. The chambers were placed in a 24-well plate and were incubated at 37°C, with 5% CO₂ for 24 h. The cells, which had not crossed the membrane were removed with a wet cotton bud. The undersides of the filters were then fixed in methanol (Sigma-Aldrich) for 10 min and stained with 0.1% crystal violent (Sigma-Aldrich), and images of the cells, which had invaded to the underside of the insert were captured. Three random fields were selected (magnification, ×40) by microscopy (CKX31; Olympus Corporation) and counted.