The ability of A549 and H1299 lung cancer cells to pass through filters coated with Matrigel® was assessed by a BD Biocoat™ invasion kit (BD Biosciences, San Jose, CA, USA); 2.5×105 of both cell lines were prepared in an aqueous environment with PBS and resuspended into each upper chamber of 6-well plates with a serum-free medium in the presence or absence of treatment; 3 mL of growth medium with 15% FBS was added to lower chambers and incubated for 20 hours. After incubation, the attached cells on the upper side of the Matrigel surface (in the upper chamber) were wiped with a cotton swab, and 50 μL of MTS reagent was added to the lower chamber. The absorbance of the media contained in the lower chamber was measured at 450 nm using the Bio-Tek ELx800 plate reader. Each experiment was carried out in triplicate.
Again, the same procedure was continued up to 20 hours of incubation in a BD Biocoat invasion kit. The cells attached to the Matrigel upper surface were then wiped using a cotton swab, and the cells invading through the Matrigel to the lower surface were fixed with 4% paraformaldehyde and stained with 2% crystal violet. The invading cells on the Matrigel lower surface were counted and photographed with the camera attached to the light microscope. The data were shown in pictures as stained cells attached to the bottom of Matrigel surface from randomly selected areas.
Again, the same procedure was continued up to 20 hours of incubation in a BD Biocoat invasion kit. The cells attached to the Matrigel upper surface were then wiped using a cotton swab, and the cells invading through the Matrigel to the lower surface were fixed with 4% paraformaldehyde and stained with 2% crystal violet. The invading cells on the Matrigel lower surface were counted and photographed with the camera attached to the light microscope. The data were shown in pictures as stained cells attached to the bottom of Matrigel surface from randomly selected areas.