Two chamber transwell assay

Migration and invasion were determined by a two-chamber transwell assay (Corning Incorporated; Corning, NY, USA). The upper side of the polycarbonate film was left untreated (migration) prior to cell seeding or was covered with Matrigel™ (500 ng/μL; BD Biosciences; Franklin Lakes, NJ, USA) (invasion, chemotaxis assay). 600 μL of complete medium was added into the lower chamber. After succinate addition, 2 × 10⁴ cells were suspended in 100 μL supplement-free medium and added into the upper chamber (inserts). After incubation for 24 h at 37  °C, transwells were gently picked up, invaded cells on the bottom of the inserts were rinsed with PBS. Nucleic acids were stained with 0.05% crystal violet and photographed with a binocular loop (Leica; Wetzlar, Germany) microscope equipped with a Mrc camera (Zeiss; Oberkochen, Germany). Cells were counted using the ImageJ software (NIH; Bethesda, MD, USA).

The migration and invasion of A549 cells were determined by two chamber transwell assay (Corning Incorporation, New York, NY, USA). Briefly, after relevant treatment or transfection, 1 × 10³ A549 cells were suspended in 200 μl serum-free RPMI-1640 medium and added into the upper chamber. 600 μl complete RPMI-1640 medium was added into the lower chamber. After incubation for 48 h at 37 °C, cells were fixed with methanol immediately. Non-traversed cells in upper chamber were removed using cotton swab carefully and traversed cells in lower chamber was stained using crystal violet and counted under microscope. Cell migration (%) was calculated by average migrated cells in propofol treatment group/average migrated cells in control group × 100%.
Cell invasion was conducted similarly with the cell migration assay except that the upper side of the polycarbonate film was spread with Matrigel (500 ng/μL; BD Biosciences, Franklin Lakes, NJ, USA). Cell invasion (%) was calculated by average invaded cells in propofol treatment group/average invaded cells in control group × 100%.