Transwell migration and invasion assay
For
evaluation of cell migration, 8-mm pore size culture inserts (Transwell;
Costar, High Wycombe, UK) were placed into the wells of 24-well culture
plates, separating the upper and the lower chambers. In the upper
chamber, 5 × 104 cells suspended in 300 μL of
serum-free medium containing different concentrations of icariin were
added, and 500 μL of cultured medium containing 10% FBS was added to
the lower chamber. For Matrigel invasion assays, 1 ×
105 cells were added to the upper chamber pre-coated
with Martrigel and containing different concentrations of icariin. After
incubation for 24 h at 37ºC, the non-migrated and non-invasive cells
located on the upper surface of the filter were removed with a cotton
swab, and the migrated and invasive cells on the bottom surface of the
membrane were fixed with methanol and stained with 0.1% crystal violet.
Finally, the cells were imaged and numbered using a microscope
(Olympus, Japan).