2.15. Transwell assay

Migration assay of the MCF-7 cells was quantified by the cells that directionally migrated through 8 µm pore size polycarbonate filter of the transwell chamber (Corning Inc., NY, USA) [44]. The MCF-7 cells of each group were suspended with serum-free RPMI-1640 containing 0.1% BSA. The cells (100 µl, 2 × 10⁵/ml) were seeded in the upper chamber, while RPMI-1640 medium containing 10% FBS (600 µl) was added into the lower chamber. After incubated for 24 h, the culture medium was discarded from the transwell chamber, then the non-migrating cells were carefully wiped from the upper chamber, and the upper chamber was washed twice with PBS. Then the cell was fixed, stained, and washed by PBS. After air-dried naturally, the transwell chamber was turned upside down and placed on the slide under a fluorescent microscopy (DM6000B, Leica, Wetzlar, Germany). Five visual fields were randomly selected for photography (× 200). The invasion test was carried out, except that the upper surface of the filters was covered with matrigel in serum-free RPMI-1640 medium at 4 °C.