2.7. In vitro analysis of cell proliferation, cell cycle, apoptosis and migration

Cells were seeded in 12‐well‐flat‐bottomed plate, with each well containing approximately 1000 cells in 900 μL of cell suspension. After a week, the cells in each well were washed twice by PBS, fixed in 100% methanol for 5 minutes at room temperature, stained with 0.1% crystal violet for 5 minutes, washed four times by PBS and finally air‐dried. For cell cycle analysis, cells were collected and fixed in 70% ethanol overnight at 4°C. Single‐cell suspensions were labelled with PI/RNase Staining Buffer (BD Pharmingen™) and analysed by flow cytometry (Beckman Coulter). Each test (1 × 10⁶ cells) was incubated with 0.5 mL buffer for 15 minutes at room temperature before analysis. For detection of apoptosis, cells were stained with Annexin V and propidium iodide using the Annexin V, 633 kit (DojinDo) according to the manufacturer's protocol, and the percentage of apoptotic cells was determined by flow cytometry (Beckman Coulter).

For migration assay, the transwell inserts were placed in a 24‐well plate with about 1 × 10⁴ cells in serum‐free DMEM media per insert. The inserts were incubated at 37°C for 12 hours in wells containing DMEM media supplemented with 10% FBS. After 12 hours, the inserts were washed with PBS, fixed with methanol for 2 minutes and stained with 0.1% crystal violet for 30 minutes. The cells on the apical side of each insert were then scraped off with a cotton swab. The number of cells that had migrated to the basal side of the membrane was visualized with a microscope at 200× magnification.