8 μm transwell insert

We seeded 5 × 10⁴ tumor cells (CAL-27 and SCC-15) into 200-μL serum-free RPMI-1640 in 8-μm transwell inserts (for migration assay, Corning, USA) or Matrigel-coated 8-μm transwell inserts (for invasion assay, Corning) of 24-well plates. RPMI-1640 medium containing 5% FBS was placed into the lower chamber. After 24 h of incubation, the cells in the upper chamber were removed with cotton swabs. Migrated cells were fixed using 4% formaldehyde and stained with 1% crystal violet. The number of cells that penetrated the membrane was counted in five random fields (400 × magnification).

Migration assay was performed using 8 μm transwell inserts (Corning, NY, USA). 1 × 10⁵ colorectal cancer cells in 100 μl serum-free medium was added into the inserts, and the inserts were then placed in 24-well plate that fulfilled with 600 μl complete growth medium. After 24 h, the inserts were washed with PBS, fixed with 4% formaldehyde, and stained with 0.5% crystal violet. Five random fields were selected for image per filter-bottom surface.

The migration and invasion capacity of tumor cells were determined by wound healing assay and transwell system using 24-well plates with 8-μm transwell inserts (Corning, Inc., Corning, NY, USA). See the Supplementary Materials and Methods for more details.

To perform cell migrating tests, this study employed 24-well plates and 8 μm Transwell inserts (Corning Life Science, MA, USA). First, THP-1 cells in 800 μl were inoculated in the lower chamber by using PMA 100 ng/ml for 24 ~ 48 h and TU212 cells or TU177 cells (105/ml) suspended in 200 μl serum-free medium were seeded in the upper chamber. After being cultured for 24 h-48 h, cells were then stained using 0.1% crystal violet and then counted. This study determined the migrated cell number with Six visual fields selected in a random manner.

Cell migration and invasion assays were performed using 24-well plates and 8 μm transwell inserts (Corning Life Science, Acton, MA, USA). For migration assays, tumor cells were suspended in 200 μl serum-free RPMI-1640 medium (4 × 10⁴ cells) containing either 2% BSA or 0.1 mM PA and cultured in the upper chamber. Fetal bovine serum-conditioned medium (10%) (700 μl) was added to the lower 24-well plates. For invasion assays, the inserts were coated with Matrigel (50 μl/well) (BD Biosciences) and kept in a humidified incubator at 37 °C with 5% CO₂ for at least 2 h before adding the cells, tumor cells were suspended in 200 μl serum-free RPMI-1640 medium (1 × 10⁵ cells) containing either 2% BSA or 0.1 mM PA and cultured in the upper chamber. After a period of culture, tumor cells remaining in the upper side of the inserts were removed with cotton swabs. Tumor cells that migrated to the lower side of the inserts were fixed in methanol and stained with 0.5% crystal violet for 30 min at room temperature. Migrated cells were photographed using Nikon Digital Sight DS-U2 (Nikon, Tokyo, Japan) and Olympus BX50 microscopes (Olympus Optical Co. Ltd., Tokyo, Japan). Five visual fields were randomly chosen to calculate the number of migrated cells.