24 well matrigel coated chambers

Transwell invasion experiments were performed with 24‐well matrigel‐coated chambers from BD Biosciences (Bedford, MA). OVCAR3 cells infected with lentivirus were seeded into the upper chambers at the density of 5 × 10⁴ cells in 200 μL serum‐free RPMI 1640 medium. The lower chambers were filled with 500 μL RPMI 1640 medium containing 10% FBS. After 24 h of incubation, noninvading cells on the top of the membrane were removed by scraping. Invaded cells on the bottom of the membrane were fixed with 100% methanol for 2 min, followed by staining with 0.05% crystal violet. The invaded cells on the membrane were then counted under a microscope. The invaded cells were collected and lysed in 500 μL lysis buffer (Bedford). Then, absorbance (OD, optical density) of invaded cell lysates was read at 570 nm. Invasion studies were repeated three times.

Cell migration experiments were performed using 24-well matrigel-coated chambers (BD Biosciences, Bedford, MA, USA), containing an 8-μm-pore-sized membrane with a thin layer of Matrigel. MCF-7 and MDA- 231 cells were transfected and harvested as mentioned above, and were seeded at a density of 2 × 10⁴ per well on the upper chamber with serum-free DMEM or L-15. Complete medium was added into the lower compartment as a chemo-attractant. The cells were allowed to migrate for 8 hours. The remaining cells were scraped out from the upper face of the filters by cotton swabs. The Matrigel membranes were fixed with ice-cold methanol and stained with 0.1% crystal violet solution. Images of three different 20× fields of view were captured from each membrane. The number of invading cells was counted. All experiments were performed in triplicate and repeated twice.