The Matrigel invasion assays were carried out in 24 well plates with 8 μm polycarbonate nucleopore filters (Corning, Tewksbury, MA, USA). The membrane for the invasion assay was covered with 100 μL BD Matrigel (diluted 1:4 with serum-free medium) in advance. The stable cells lines treated with or without tocilizumab (5 μg/mL, Selleck Chemicals) were seeded in the upper chambers; the lower chambers were filled with medium containing 10% FBS along with or without 20 ng/mL IL-6. In addition, the lower chambers were also filled with TAM cells supernatants along with or without neutralizing antibodies to IL-6 (anti-IL-6; R&D Systems). After a 48 h incubation, the cells adhering to the lower filter surface were counted.
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