24 well modified boyden chamber

The migration of HUVECs was investigated using a 24-well modified Boyden chamber (8 μm, Corning). After treatment with PA and/or recombinant human omentin-1 (concentration as mentioned above) for 24 h, HUVECs were collected and resuspended in serum-free DMEM. Approximately 5 × 10⁴ cells in 200 μL of serum-free DMEM were seeded in the upper chamber, while the lower chamber was filled with 600 μL DMEM containing 10% FBS. After 24 h incubation, the cells on the upper face of the membrane were wiped, while those that migrated to the lower face of the membrane were fixed with 4% paraformaldehyde for 20 min and stained with crystal violet solution (C0121, Beyotime) for 5 min. Images were obtained using a microscope (Leica DM5000 B). The number of migrated HUVECs was counted in three random fields (200× magnification).

The migratory and invasive potential of gastric cancer cells was assessed by using 24-well modified Boyden chambers (Corning, USA) with the polyethylene terephthalate membranes either uncoated or precoated with diluted Matrigel matrix (BD Biosciences, USA). After the appropriate treatments, MKN45 or BGC823 cell suspensions (1 × 10⁵ cells/well) in 200 μL of serum-free medium were transferred and cultured in each upper insert. Meanwhile, medium containing 10% FBS (500 μL) was applied to the lower compartment to induce migration or invasion in 24-well plates. After 24 h, Non-migrating or non-invading cells on the upper chambers were removed, whereas cells that had migrated or invaded through the lower side of the inserts were fixed in paraformaldehyde, rinsed with distilled water, stained with crystal violet, counted, and photographed under an inverted microscope (Nikon, Japan).