The tumor invasion assay was performed using a modified Boyden chamber (Neuro Probe, Cabin John, MD) as described elsewhere [65 (link)]. Matrigel (Collaborative Biomedical Products, Bedford, MA) was diluted with cold filtered distilled water to receive the concentration 25 μg/50 μl and applied to the upper to 8 μm pore size polycarbonate membrane filters of the upper well. Briefly, U2OS and HOS cells were treated with indicated concentrations (0, 25, 50, 75, 100 μM) of nobiletin for 24 h. The migrated cells passed through the basement membrane layer and clung to the bottom of the Boyden chamber membrane, while non-invasive cells stayed in the upper chamber. The data were presented as the average number of cells attached to the bottom surface from randomly chosen files under a light microscope. In order to measure the ability of osteosarcoma cells on migration, cells were seeded into the upper well of Boyden chamber which were not coated with Matrigel. Migration of cells treated with different concentrations of nobiletin was measured as described in the cell invasion assay.
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