Assessing Cell Invasiveness via Transwell

The invasiveness of HeyA8, HeyA8-MDR, and SKOV3IP1 cells was determined as previously described [83 (link)]. Briefly, cells were treated with 50 nM of the indicated oligonucleotide, and 48 hours later, live cells were collected and washed with serum-free media. Fixed numbers of the viable cells (4 × 10⁵ cells), were resuspended in 1 mL of serum-free RPMI-1640 medium and added onto 6-well plate Transwell inserts (8-μm-pore size; Fisher Scientific) coated with a Matrigel basement membrane (0.7 mg/mL; BD Biosciences). Lower chambers were filled with 2 mL of medium supplemented with 15% FBS. After 24 hours, non-invading cells on the upper surface of the filter were removed with cotton swabs. Cells that invaded through the Matrigel onto the lower side of the filter were fixed, stained with the Hema-3 Stain System (Fisher Scientific), and photographed. The number of cells that invaded the lower side of the filter was counted in 5 fields and expressed as the mean number of cells from triplicate measurements.