Costar transwell cell culture chamber inserts

The effects of STAT1 and STAT1-CC on migration and invasive ability of lung cancer cells in vitro were examined using transwell assays. Transwell assays were performed in Costar transwell cell culture chamber inserts with an 8 μm pore size, placed in a 24-well cell culture plate (Corning Costar Corporation, Cambridge, MA, USA) [14 (link)]. Biological triplicates were performed for each of these assays.
For the cell migration assay, cells (1 × 10⁵) were suspended in serum-free medium and seeded to the upper part of chamber. Complete medium with 10 % FBS was added to the lower chamber as a chemoattractant. After 24 h, the transwell membrane was fixed with 4 % paraformaldehyde, and stained with 0.1 % cresyl violet. Migrated cells were counted under a microscope at 400× magnification. For the cell invasion assay, cells (1 × 10⁵) were suspended in serum-free medium and seeded into the upper compartment of the transwell chamber coated with 10 μl of diluted Matrigel (BD Biosciences). Complete medium with 10 % FBS was added to the lower chamber. After 48 h, cells were fixed, stained, and counted.

Migration assay was performed using Costar transwell cell culture chamber inserts (Corning Costar Corporation) with an 8 µm pore size. Briefly, 5 × 10⁴ RAW 264.7 cells or human osteoclasts from buffy coat were placed in the upper chamber in 200 µl of DMEM supplemented with 10% FBS or α‐MEM supplemented with 10% FCS, respectively. Standard medium with 1% FBS or FCS and containing 20% of each MLO‐Y4 cell‐conditioned medium for 18 h were placed in the lower compartment. Furthermore, in others experiments, 2 µg/ml of neutralizing antibody anti‐mCXCL5 (Thermo Fisher Scientific) and 1 µg/ml of anti‐mIL‐6 (R&D Systems) were used. Then, the medium and the upper cell layer were removed with the aid of a cotton swab, and cells on the underside of the transwell were fixed with PFA 4%, stained with crystal violet (Sigma Aldrich) and counted in five randomly selected fields at X200 magnification using Leica DM 5500B microscope.