|Incubate at 37°C in a humidified atmosphere of 5% CO2 for 48 h
Cell invasion was assayed using the QCM ECMatrix Cell invasion assay (Millipore) using polycarbonate membranes with 8-μm pores and a layer of reconstituted basement membrane matrix. Cells (3 × 105) were suspended in 300 μL serum-free medium and carefully transferred to the upper chambers of the devices. The lower chambers were filled with 500 μL medium containing 10% FBS as an attractant, and the chamber was incubated at 37°C in a humidified atmosphere of 5% CO2 for 48 h. Then, non-invading cells were removed from the upper chamber with a cotton swab. Cells that had invaded the polycarbonate membrane to the lower surface were fixed, stained with crystal violet, and quantified by counting five random microscope fields at a magnification of 400×. Full paper
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