Cls3422

Before migration, cells were serum-starved overnight. Then, 2 × 10⁵ OS cells or 1 × 10⁶ HUVECs in 200 μl of the serum-free RPMI 1640 medium were seeded to each top well of a 12-well Transwell Boyden system (8 μm pore size, Sigma, CLS3422), and 500 μl RPMI 1640 medium supplemented with 10% fetal calf serum was added to the lower chamber. Cells were allowed to migrate for 24 h at 37 °C, in 5% CO₂. After removing cells on the upper surface of the filter using cotton swabs, cells that invaded through the membrane were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet solution for 15–20 min. The number of cells that reached the lower part of the Transwell filter membrane was counted with Image J software (National Institutes of Health, USA) and plotted as the number of cells per optic field (× 200). Experiments were carried out in triplicate.
The invasive procedure is almost the same as the migration assay except for the 8 μm pore size (corning, CLS3422) coated with 150 μg of Matrigel (65 μl of 2.3 mg/ml of Matrigel, Becton Dickinson Matrigel Basement membrane Matrix, phenol-red free, Collaborative Research Cat. no. 40234C) used.

The upper chamber of the Transwell (8-μm-pore, CLS3422, Sigma–Aldrich, U.S.A.) was pre-coated by 50 μl Matrigel (M8370; Solarbio, Beijing, China), and contained the transfected MCF7 cells at 37°C with 5% CO₂, while 500 μl RPMI-1640 with 10% FBS was added into the lower chamber as chemoattractant. After 24 h, the lower chamber was washed with PBS multiple times. The cells were then fixed by paraformaldehyde solution (4%) for 30 min and then stained by 0.1% Giemsa (G4640, Solarbio, China) for 20 min. Cell number was counted from five random fields under a stereo microscope (SZX10; Olympus, China) and photographed at 250× magnification.

The invasion assays were carried out following a modification of the protocol described in Hemberger et al., 2004 (link). The Transwell filters (Sigma, CLS3422) were coated with 100 μl of a 1:20 dilution of cold Matrigel (Corning 356231) in RPMI 1640 medium. The Matrigel layer was allowed to dry overnight at room temperature and was rehydrated the next day with 100 μl of supplemented RPMI 1640 medium for 2 hr at 37°C under 95% humidity and 5% CO₂. Confluent 60 mm dishes of TS cells were trypsinized and resuspended in RPMI at 10⁶ cells/ml; 100 μl of this cell suspension (10⁵ cells) was added to the top chamber, and the bottom chamber was filled with 800 μl of culture medium.
After the specific times of incubation, Transwell inserts were fixed for 5 min in 4% PFA and washed with 1× PBS. Cells that remained on top of the filters as well as the Matrigel coating were scraped off. Filters were stained overnight with hematoxylin and excised under a dissecting microscope, removing all residual cells from the top of the filters. Filters were mounted with 20% glycerol in 1× PBS and photographs of each filter were quantified with ImageJ.