8 μm chamber inserts

The experiment was performed in 24-well Transwell plates with 8 μM chamber inserts (Corning, USA). Forty-eight hours posttransfection, cells were plated into the upper chamber in 100 μL serum-free medium (3 × 10⁵). 600 μL of fresh complete medium was added to the lower chamber of a 24-well plate. The cells were incubated for 48 h, after which the top membrane surface cells were removed by wiping. Cells that had invaded the lower chamber were fixed using paraformaldehyde for 30 min, stained with 0.1% crystal violet for 20 min, imaged, and counted under a microscope.

Transwell assay was performed by using 24-well plates with 8-μm chamber inserts (Corning Life Science, USA) as previously described [15 (link)]. The inserts were precoated with Matrigel (50 μl/well, BD, USA) and 2 × 10⁵ cells were seeded in the upper chamber with serum free medium in triplicate. Medium containing 10% FBS was added to the lower chamber as chemo-attractant. After incubation for 24 h, the cells above the Matrigel layer were removed by cotton swab, and the cells below the membrane were fixed by methanol, stained with 0.1% crystal violet for 10 min, and counted from five randomly chosen fields for each well.

To perform the wound healing assay, cells were seeded into 6-well plates and allowed to grow to a more than 90% confluency. The monolayer was then uniformly wounded using a 200-μL pipette tip. After washing with fresh medium, the cells were incubated in a serum-free medium for 24 h, and images were taken from 5 random fields of view using a microscope. The wound closure percentage was evaluated by comparing the changes in the wound area before and after 24 h.
For the Transwell migration assay, 24-well plates with 8 μm chamber inserts (Corning Life Science) were used. A total of 1 × 10⁵ cells were seeded in the upper chamber with a serum-free medium in triplicate. Medium containing 10% FBS was added to the lower chamber as a chemo-attractant. After incubation for 24 h, the cells in the chamber were removed by cotton swabs, and the cells below the membrane were fixed with 4% PFA, stained with 0.1% crystal violet for 10 min, and counted.