Matrigel coated transwell chamber

To measure cell migration, transwell inserts with 8-μm pore sizes were placed in 24-well plates. HepG2 and Hep-3B cells were treated with 20 μM arctigenin for 2 days. Then, 1 × 10⁵ HepG2 and Hep-3B cells were seeded in the upper chamber. The lower chamber was filled with Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS). Twenty-four hours later, cells migrating to the underside of the transwell membrane were fixed with 4% paraformaldehyde and stained with crystal violet. The migratory cells were viewed with a NIKON microscope and their numbers were counted in five different fields of view (Han et al., 2017 (link)). Matrigel-coated transwell chambers (BD Biosciences, United States) were used to detect cell invasion. HepG2 and Hep3B cells were seeded on the matrigel matrix in the upper chamber. DMEM culture medium was added to the bottom chamber with 10 nM of arctigenin. After 48 h, the cells that had invaded the matrigel-coated membranes were fixed with paraformaldehyde and stained with crystal violet (Zhang et al., 2015 (link)).

Invasive capacity of LNCaP and PC-3 cells was investigated with Matrigel-Coated Transwell Chambers (BD, United States ). Cells were planted onto the upper chamber. Following overnight incubation, cells in the lower chamber were fixed by 4% paraformaldehyde as well as stained by 0.1% crystal violet. Invasive cells were counted under a microscope (Olympus, Japan).

Cells were initially transfected with 10 nM pre-miR-138, 10 nM pre-miR-scrambled control, 50 nM Locked Nucleic Acid (LNA) miR-138, or 50 nM LNA-scrambled control. After 48 hours, cells were trypsinized, then 1.5×10⁵ cells from each condition were seeded inside the Control or Matrigel coated trans-well chambers (BD Bioscience, Franklin Lakes, NJ) with RPMI 1640 medium containing 1% bovine serum. The chambers were then placed in 24-well plates containing RPMI 1640 medium, and 20% bovine serum served as the chemo-attractant for the cells. After incubating for 24 hours, cells were fixed and stained using the Diff Quik® Set (Siemens Healthcare Diagnostics Inc., Deerfield, IL); then the number of cells that invaded to the opposite side of the membrane was counted under a microscope. Results were analyzed to derive each condition's invasion index, which represented the invasive ability of the cells over their migration ability, calculated as (Invasion/Migration of Test Cell)/(Invasion/Migration of Control Cell) for each condition. This analytical method thereby accounted for any cytotoxic effects from pre-miR or LNA transfections.