Boyden chambers with 8 m pore size polycarbonate membranes

The cells were first transfected with miR-138-5p/miR-204-5p mimics, inhibitors, the EGFR overexpression lentivirus, EGFR siRNA and the relevant negative control. Twenty-four-well Boyden chambers with 8-µm pore size polycarbonate membranes (Corning Inc., Corning, NY, USA) were used and ~10⁵ cells were seeded onto the upper chamber with 200 µl of serum-free medium at 24 h after transfection. Approximately 600 µl of medium supplemented with 10% serum was added to the lower chamber as a chemoattractant. Twenty-four hours after incubation, the non-migrating cells on the upper surface of the membrane were gently scraped off with cotton swabs. Subsequently, the membranes were fixed using methanol and stained with a three-step staining set (Thermo Fisher Scientific, Inc., Paisley, UK). The migrating cells were calculated in five visual fields randomly selected from each membrane. All experiments were performed in triplicate. The data of experimental group and control group were input to statistical analysis.

Tumor cell invasion was analyzed in 24-well Boyden chambers with 8-µm pore size polycarbonate membranes (Corning Incorporated, Corning, NY, USA). The membranes were coated with 60 µg of Matrigel (cat. no. 3432-005-01; R&D Systems, Inc., Minneapolis, MN, USA) to form the matrix barrier. Pancreatic cancer cells transfected with NC or si-MIF were resuspended in 100 µl serum-free DMEM 36 h post-transfection, and were added to the upper compartments of the chambers. The lower compartments were filled with 600 µl DMEM or RPMI-1640 with 10% FBS. Following an incubation at 37°C for 24 h, the cells remaining on the upper surfaces of the membrane that had not invaded through the matrix were removed. The invaded cells on the lower surfaces of the membrane were fixed with 100% methanol at room temperature for 10 min, stained with 0.1% crystal violet at room temperature for 15 min and counted under a light microscope at ×400.