Transwell Invasion Assay
Cell invasion assay was performed using transwell chambers with polycarbonate filters (8.0 μm pore size; Costar, USA) in 24-well plates. In brief, the upper transwell chambers were precoated with 35 μL Matrigel (BD Biosciences, USA) and serum-free DMEM medium mixtures at a ratio of 1:9. Before the experiment, TC was pretreated with different concentrations (0, 6, 12 μg/mL) for 48 h. Then 2.5 × 105 of AMDC was resuspended in 300 μL serum-free DMEM and plated on the upper side of the filter, while 600 μL complete DMEM f12 containing 10% UY was placed in the lower plate. Cells were incubated at 37 °C in a 5% CO2 for 48 h. The invaded cells in the lower chamber were fixed with 4% paraformaldehyde for 25 min and stained with 0.1% crystal violet (Beyotime, Beijing, China) for 30 min at room temperature, after which non-invasive cells on the upper surface of the membrane were gently removed with a cotton swab. Dry at room temperature for 24h. Next the microscope (Nikon, Japan) was employed to capture the images of the cells invaded to the lower chamber. Three fields per filter were randomly selected under the microscope to count the invaded cell numbers.