2.6. Transwell assays

Transwell assays were performed to explore cell migration and invasion. For cell invasion, Matrigel was diluted with medium six times after overnight thawing and then put into the upper chamber. Cells (1×105/well) were seeded into the upper chamber pre-coated with Matrigel and to the bottom chamber 500 μL complete medium was added . After 24 h incubation, cells that did not invade were removed by cotton swabs while invasive cells were fixed using 4% paraformaldehyde for 30 min, stained by 0.1% crystal violet for 20min. Next, PBS was used to wash off the floating color and the status was imaged under an optical microscope. Cells were calculated in several random fields. For cell migration, the density was 5, 000 cells per well and the chamber without Matrigel was utilized. The experiment was repeated at least three times and all the results were present as mean ± standard deviation (SD).