8 mm transwell

A transwell assay was also used to assess cell migration. Initially, 2 × 10⁴ cells seeded in the upper chambers of every 8 mm transwell (Millipore, Billerica, MA, United States) were irradiated with 4 Gy of X-rays. The cells in the upper chamber were incubated in serum-free medium, whereas medium with 10% serum was added to the lower chamber. After removing cells on the upper surface of the filter after 48 h, cells attached to the lower chamber were fixed with 4% paraformaldehyde (Biosharp) for 30 min, stained with 0.1% crystal violet solution (Biosharp)for 30 min. Three areas were selected randomly for photography and calculation using an inverted microscope (Carl Zeiss).

The cell invasiveness was examined with the 8-mm transwell (Millipore, Billerica, MA, USA)
with (invasion assays) or without (migration assays) the coating of Matrigel. According to previous study [39 (link)], the cells were seeded into serum-free medium and added to the upper chamber (1 × 10⁴ cells per chamber) coated with 200 mg/mL of Matrigel. A medium containing 10% FBS was added to the lower chamber as a chemical attractant. After 24 h of incubation, the cells in the upper chamber were removed by wiping with a cotton swab. We then fixed the cells invading the lower surface of the filter in 70% ethanol for 30 min, and stained them for an additional 10 min with 0.2% crystal violet. The invading cells were photographed and counted across five random fields of view in each cell under an optical microscope at 200× magnification.