*4.5. Cell Invasion Assays*

The 24-well plate Transwell inserts (pore size 8 µm, translucent) (Greiner bio-one, Stonehouse, UK) were coated with 30 µL of ice-cold Matrigel (Corning, VWR, Lutterworth, UK) mixed with cold serum-free medium at a ratio of 1:3. The coated Transwell inserts were left to set at 37 ◦C for 2 h. Fifty thousand cells were seeded in each Transwell insert suspended in 300 µL of 0.5% *v*/*v* FBS culture medium. Eight hundred microliters of chemoattractant (10% *v*/*v* FBS cell culture medium) was added to the wells, and the Transwell inserts were placed in each of the wells that contain a chemoattractant. The Transwell inserts were incubated for 24 h at 37 ◦C. After 24 h, the culture medium in the Transwell inserts were discarded, and a cotton swab moistened with Dulbecco's phosphate-buffered saline (DPBS) (Biowest) was used to, gently but putting firm pressure, rub the inside of the Transwell inserts to remove cells, and this process was repeated with a second cotton swab. The cells that moved to the lower surface of the membrane of the Transwell inserts were stained with Differential Quik stain (Modified Giemsa) (Generon, Slough, UK), following the manufacturer's instructions. The Transwell inserts were then rinsed twice with water. A cotton swab moistened with DPBS was used to gently but putting firm pressure wipe again the inside of the Transwell inserts to rid of any cells left. The Transwell inserts were left to dry at room temperature. To determine the percentage cell invasion, the invaded cells were counted from randomly selected five fields of view for each Transwell insert under a light microscope at a magnification of ×40, and the mean number of invaded cells were obtained.
