4.8. Cell Migration Assay

The migration assay was performed using transwell chambers. T47D and BT20 cells were transfected with pontin-targeting or control siRNA oligos for 48 h. Then, transfected cells were trypsinized and resuspended at 2 × 10⁴ cells in 200 µL serum-free medium. The resuspended cells were placed into the upper chambers of a 24-well transwell plate (8 mm pore size; Corning Inc.). The lower chambers were filled with 700 µL complete medium with 10% FBS. Following incubation for 24 h at 37 °C, non-invading cells were removed from the upper chamber with a cotton swab. Invading cells on the lower surface of the inserts were fixed with 4% paraformaldehyde and stained with 1% crystal violet. Three random fields for each insert were counted at 200× magnification. Data were recorded as the mean ± SD of at least three independent experiments.