C0133

Prior to the Transwell assay, 10 mL Matrigel (M8370, Solarbio Lifesciences, China) was thawed at 4°C overnight, and diluted with chilled non-serum growth medium. Transwell chambers with 8-μm pore (CLS-3422, Sigma-Aldrich, USA) were coated with 50 μL pre-thawed Matrigel and placed on the 24-well plates that were placed in a humidified incubator at 37°C to allow gelling. Then, after being detached with Trypsin/Ethylenediaminetetraacetic acid (EDTA) (T3924, Sigma-Aldrich, USA), 1 × 10⁵ cells/well transfected parental and DDP-resistant AGS and HGC-27 cells were transferred to the upper Transwell chamber with 200 μL non-serum medium at 37°C with 5% CO₂, and 700 μL complete medium was added into the corresponding lower chamber as the chemoattractant.
48 hours later, the excessive Matrigel was removed, while the lower Transwell chamber was fixed in 4% paraformaldehyde (P0099, Beyotime Biotech, China) at room temperature for 15 minutes and stained using 0.1% Giemsa (C0133, Beyotime Biotech, China) for 20 minutes. Invaded cells were photographed in five picked fields in a random manner under an inverted optical microscope at a magnification of × 250.

Cells were washed 3 times with pre‐cooled PBS and fixed with methanol. After drying, 1x Giemsa solution (Beyotime, C0133, SH, CN) was added to stain for 2 min and then rinsed with pure water. Cells were photographed under a Nikon TE300 Inverted Fluorescence Microscope.