24 well transwell inserts with 8 μm pore membrane filters

Migration assays were performed as previously described [24 (link)]. Briefly, using 24-well transwell inserts with 8 μm pore membrane filters (Corning). For pharmacological studies, migration assays were performed 24 h after A549 cells were treated with the IKKβ inhibitor CmpdA. Alternatively, cells were transfected with siRNAs as described above and migration was analysed 72 h post-transfection. In both cases, for each well 5 × 10⁴ A549 cells were resuspended in 300 μL serum-free medium, added to the upper chamber and incubated for 24 h at 37 °C in 5% CO₂. Complete medium (500 μL) was added to the lower chamber to be used as chemoattractant. Non-migrating cells were scraped off the upper surface of the membrane with a cotton swab, and migrating cells on the bottom surface were fixed in 4% paraformaldehyde in PBS and stained with crystal violet. Images were obtained under an IX51 Inverted Microscope (Olympus, Tokyo, Japan) and cells from three random fields of view from three independent experiments were analysed using ImageJ software.