Boyden transwell chamber assay for cell migration and invasion

Corning Costar Transwell cell culture inserts (Corning Inc., Corning, NY, USA) were employed to investigate the effect of ANXA5 overexpression on the in vitro migration and invasion properties of Hca-P cells. For the migration assay, 600 µl RPMI-1640 with 20% FBS was loaded into each lower chamber. A total of 10,000 cells from each group were separately loaded into one upper chamber in 200 µl RPMI-1640 medium and incubated at 37°C with 5% CO2 for 24 h. The non-migrated cells on the upper surface of the insert were carefully wiped off using cotton swabs. The cells that had migrated to the lower surface of the filter were fixed in methanol (Sigma-Aldrich; Merck KGaA) for 30 min, dried for 5 min at room temperature, stained in 0.1% crystal violet for 40 min, washed with PBS (200 µl) and counted by randomly selecting 5 fields per well with a BX63 upright light microscope (Olympus Corporation, Tokyo, Japan) at a magnification of ×200. For the invasion assay, the Transwell surface was coated with 50 µl ice-cold ECM gel (2 mg/ml; Sigma-Aldrich; Merck KGaA) and incubated at 37°C for 6 h. Subsequently, the Transwells were dried at room temperature for 30 min, and then hydrated with 50 µl serum-free RPMI-1640. RPMI-1640 (600 µl) with 20% FBS was then added into the lower chamber. Next, 7,500 cells suspended in 150 µl RPMI-1640 from each group were loaded into the upper chamber. The rest of the steps were the same as those described for the migration assay.