Transwell permeable support polycarbonate membrane inserts

The effect of scutellarin on BV-2 microglial cell migration was assessed using Transwell™ permeable support polycarbonate membrane inserts (8.0 μm pore size; Costar, USA), placed in a 24-well plate. After treatment with scutellarin at different concentrations (0.54 mM, 0.27 mM and 0.11 mM) for 3 h, the cells were washed with PBS, trypsinized, and resuspended in basic medium. The 200-μl cell suspension without FCS (3 × 10⁴ cells) was seeded into the inside of the insert, and 600 μl DMEM with 20% FCS was added into each well of the 24-well plate. After incubation at 37°C with 5% CO₂ for 20 h, the medium was discarded and the cells remaining inside the insert were carefully removed. Cells were then fixed with 100% methanol for 15 min at room temperature and were stained with 0.5% of crystal violet at room temperature for 30 min. The cells were rinsed with distilled water until excess dye was removed. The images of migrated cells on the lower surface of the insert were captured at a magnification of 120x. Cells in five visual fields were counted on each insert using Image J software. Results are expressed as mean (± SD) of the number of cells per visual field.