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.