Chamber insert

Matrigel matrix (BD Bioscience, Franklin Lakes, NJ) was diluted with cold phosphate-buffered saline (PBS, 1:4, v/v). Then, 100 μL diluted Matrigel was coated on the chamber insert (pore size: 8 μm; Corning, Corning, NY). A density of 1 × 10⁵ cells was seeded on top of the Matrigel matrix in serum-free DMEM, and the receiving chamber was filled with DMEM containing FBS. At 36 h post-seeding, the Matrigel matrix with non-invading cells was removed with cotton swabs. Cells at the basolateral membrane were fixed in 4% paraformaldehyde followed by staining with crystal violet. Images of invaded cells were taken after washing and air-drying. All experiments were conducted in triplicate.

For measuring cell migration, KP3L and PANC-1 cells were cultured in 6-well plate until full coverage. A wound was created by scraping cell layer using a 10 μL pipet tip. Images were captured at 0 and 48 h to observe the wound closure by cell migration. For measuring cell invasion, 1 × 10⁵ KP3L and PANC-1 in serum-free medium was seeded onto the chamber insert (Corning, Corning, NY, USA) coated with Matrigel Matrix (1:3 v/v in cold PBS, BD Bioscience). The received chamber was filled with medium containing FBS. Forty-eight hours after cell seeding, cells attached at the basolateral membrane of chamber insert were fixed and stained with crystal violet. Images were captured under microscope (200× magnification, Leica).

To analyze the invasion and migration ability of cells, scratch tests and transwell migration assays were performed according to published methods. For the cell scratch test [26 (link)], cells were digested into a single cell suspension, inoculated into a 6-well plate at a concentration of 10⁶ cells/well overnight, and scratched vertically with a 100 μL micropipette tip the next day. Thereafter, the cells were washed twice with PBS and placed in a serum-free culture medium. After 12 h, cells were counted under an inverted phase-contrast microscope in five random fields. Each migration test was run in triplicate. The wound area was photographed immediately at 0 (W0) and 12 h (W12) and the relative invaded rate in each group was calculated as the following formula (1): $\mathrm{Relative} \mathrm{invaded} \mathrm{rate} \left(\%\right)=\frac{\mathrm{W}0-\mathrm{W}12}{\mathrm{W}0}\times 100\%$
Transwell migration assays [27 (link)] were performed according to standard protocols. 786-O cells (1 × 10⁵ cells/mL in 0.2 mL) were seeded in each chamber insert (8 μm pore size, Corning) of a 12-well plate. After 12 h of incubation, cells in the transwell chamber were fixed with 4% formaldehyde in 1× PBS and stained with 3% crystal violet staining buffer. The fixed crystal violet in each chamber insert was dissolved in 500 μL of 2% acetic acid. Absorbance (A) of the cleanout fluid was determined using a microplate reader (Infinite M1000, Tecan, Switzerland) at 570 nm.