A Transwell chamber with a porous membrane (Corning, catalog no. 3422) was placed in a 24-well plate.
Then, the same number (1×10 5 ) of A549 or H1299 cells in 200 µl of serum-free culture medium was added to the upper chamber, and the lower chamber contained 500 µl of growth medium containing 10% FBS. After 12 h, nonmigrated cells on the top of the porous membrane were removed by a cotton swab, and the migrated A549 or H1299 cells on the bottom surface were xed (4% paraformaldehyde) and stained with 0.5% crystal violet. The cells that passed though the membrane were visualized by the size of the colored area. Each experiment was repeated 3 times.
For the invasion assay, the Matrigel (Corning, catalog no. 354234) was diluted (1:8) with PBS and poured into chambers above which were placed in 24-well plates. Then, 1×10 6 cells were placed on the Matrigel, and the lower chamber contained 500 µl of growth medium containing 10% FBS. The cells were cultured at 37℃. Then, 24 h later, the cells that passed through the Matrigel and porous membrane were visualized with the same protocol as in the migration assay. Each experiment was repeated 3 times. OLYMPUS SZX10 microscope (8×) and Image J software was used to analyzed cell number.
Then, the same number (1×10 5 ) of A549 or H1299 cells in 200 µl of serum-free culture medium was added to the upper chamber, and the lower chamber contained 500 µl of growth medium containing 10% FBS. After 12 h, nonmigrated cells on the top of the porous membrane were removed by a cotton swab, and the migrated A549 or H1299 cells on the bottom surface were xed (4% paraformaldehyde) and stained with 0.5% crystal violet. The cells that passed though the membrane were visualized by the size of the colored area. Each experiment was repeated 3 times.
For the invasion assay, the Matrigel (Corning, catalog no. 354234) was diluted (1:8) with PBS and poured into chambers above which were placed in 24-well plates. Then, 1×10 6 cells were placed on the Matrigel, and the lower chamber contained 500 µl of growth medium containing 10% FBS. The cells were cultured at 37℃. Then, 24 h later, the cells that passed through the Matrigel and porous membrane were visualized with the same protocol as in the migration assay. Each experiment was repeated 3 times. OLYMPUS SZX10 microscope (8×) and Image J software was used to analyzed cell number.