Porous polycarbonate membrane 8.0 μm

A migration assay was performed by using 24-well culture inserts with a porous polycarbonate membrane (8.0 μm, Millipore, Billerica, MA). For the Matrigel invasion assay, the filters were pre-coated with 30 μl of Matrigel (BD Biosciences, USA) for 3 hours. The migration and invasion assays were performed according to our previously published protocol [42 (link)]. In brief, 2 × 10⁴ cells in 200 μl of serum-free medium were added to the upper chamber, and 800 μl of medium with 5% serum was placed in the lower chamber. The plates were incubated for 24 hours at 37°C in 5% CO₂. Cells that did not migrate or invade through the pores were removed with a cotton swab. Cells on the lower surface of the membrane were examined and counted under a microscope. Each experiment was repeated at least three times.

In the transwell migration assay, 24-well culture inserts with porous polycarbonate membrane (8.0μm, Millipore) were used, and in the matrigel invasion assay, 30μl matrigel (BD Biosciences) was evenly smeared on the bottom of the upper chamber of transwell insert and incubated for 3 hours to be ready for use. Then 2 × 10⁵ cells were added into the upper chamber and 5% serum medium 800μl was added into the lower chamber. After conventional culture for 24 hours, the transwell insert was taken out. The endothelial cells and microglia on the chambers were wiped out with a cotton swab. 4% raformaldehyde was added for fixation. Crystal violet was added and the cells were incubated for 2 minutes for staining. Then PBS was added to turn the cells blue. The microscope was set at a magnification of 200x and 5 fields of view were chosen for which the mean value was calculated.