Cell culture chamber

Cell migration and invasion were detected by the Transwell method. Cells were collected with 0.25% trypsin-EDTA solution (Solarbio, China) and centrifuged. Cells were resuspended at a density of 5 × 10⁵ cells/ mL in RPMI-1640 medium and treated with resveratrol at different concentrations (0, 12.5, 25, and 50 μM). Then, 100 μL of treated cells was inoculated into the upper compartment of the cell culture chamber (Corning, United States), and 700 μL of medium containing 10% FBS was added to the lower compartment for further culture for 24 h at 37 °C. After incubation, the cells on the upper surface of the membrane were removed. Cells below the membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet ammonium oxalate solution. In the cell invasion assay, the Transwell chamber was coated with 1 mg/ mL Matrigel (Corning, United States) before the experiment, and the other steps were basically the same as in the migration assay. Cell migration and invasion were photographed with an inverted microscope.

To detect cell migration, we used cell culture chambers according to the manufacturer’s instructions (Corning Costar Corp., Cambridge, MA, USA), we first used trypsin digested cells and then collected cells with medium containing 10% FBS. Transfectant cells or control cells (5×10⁴ cells) (approx. 250 mL) were transferred into the upper chamber, and the lower chamber was filled with 600 mL of medium containing 10% FBS. Then, the plate was placed in the incubator for 24 h. The membrane was fixed with 4% PFA and stained with 0.4% crystal violet solution for 20 min. Matrigel invasion performed using a procedure similar to that used for the migration assay. Cell migration and invasion ability were assessed by counting the cells that had migrated through the membrane. A total of five random fields of view were selected, and images were captured by a microscope at 20× magnification.