Migration and invasion abilities of the cells were examined using the Transwell assay performed in Transwell cell culture chambers (8.0 μm pore size) 30 (link),31 (link). Briefly, cells were inoculated in a 24-well Transwell chamber for migration experiments or in 10% FBS-containing DMEM in chambers coated with Matrige for invasion experiments. A 200 μL suspension of cells was added at a density of cells/well, and 600 μL of DMEM containing 10% FBS was added to the lower chamber. After the GC cells had adhered to the chamber wall (after 6 h), the complete medium was replaced with serum-free medium, and the cells were treated with 5-FU or a combination of MLT and 5-FU for approximately 40 h. Next, 10% serum (60 μL) was added to the lower chamber and allowed to stand for approximately 8 h. The medium in the upper chamber was washed twice with preheated PBS. The cells were fixed with 4% paraformaldehyde (Biosharp, Beijing, China). Invasive cells on the back of the membrane were stained with 0.1% crystal violet (Biosharp). The cells that could not penetrate the membrane were removed using a wet cotton swab. The numbers of migrating or invading cells were calculated by counting in five random fields of view under a light microscope and expressed as mean ± SE.
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