Transwell chambers for 24 well plates

Invasion assays were performed in triplicate using Transwell chambers for 24-well plates (0.8-μm pore size; Corning, Costar) coated with ECM gel (40 μl; Sigma) according to the manufacturer's instructions. Cells (2 × 10⁴ to 6 × 10⁴) were plated in 200 μl RPMI 1640 medium with 0.1% fetal bovine serum into the upper chamber. The lower chamber was filled with 700 μl RPMI 1640 medium with 30% fetal bovine serum. After culture for 24 h to 72 h, noninvaded cells were mechanically removed by a cotton swab. The invaded cells at underside of the membrane were fixed with 4% formalin and stained with 0.1% crystal violet for visualization. Cells were counted in ten respective microscopic fields (× 40 magnification) and photographed.

Transwell chambers for 24-well plates (Corning, NY, USA) were prepared in advance, and 100 µL medium without FBS was added to the upper chamber with 5 × 10⁴ cells. Next, 600 µL medium with 30% FBS, serving as the chemoattractant, was added into the lower chamber. Cells were cultured for 44 h, and then the transmigrated cells were fixed with 4% paraformaldehyde and stained using crystal violet after removing the non-transmigrated cells. Fluorescence microscopy (Olympus, Tokyo, Japan) was used for imaging and cell counting.

Migration assays were performed in triplicate using Transwell chambers for 24-well plates (0.8-µm pore size; Corning, Costar) coated with ECM gel (40 µL; Sigma) according to the manufacturer’s instructions. BxPC-3 cells (2 × 10⁴) and PANC-1 cells (6 × 10⁴) were plated in 200 µL RPMI 1640 medium with 0.1% fetal bovine serum into the upper chamber. The lower chamber was filled with 700 µL RPMI 1640 medium with 30% fetal bovine serum. After culturing for 24–72 h, noninvaded cells were mechanically removed by a cotton swab. The invaded cells on the underside of the membrane were fixed with 4% formalin and stained with 0.1% crystal violet for visualization. Cells were counted in ten respective microscopic fields (×40 magnification) and photographed.