*4.12. Matrigel Invasion Assay*

Cell invasion was measured using Corning BioCoat Tumor Invasion System (Corning, New York, NY, USA) following manufacturer's protocol. EVC, AKI, and BKI iOTR cells were stained with CellTracker™ Green 5-chloromethylfluorescein diacetate (CMFDA) (Invitrogen, Carlsbad, CA, USA). Four replicates of each cell line were plated at a density of 10,000 cells/500 µL DMEM/F-12 (1:1) media without phenol red and fetal bovine serum, in a 24-multiwell insert plate with 8 µm pore size polyethylene terephthalate membrane coated with uniform layer of matrigel matrix. DMEM/F-12 (1:1) media without any cells was added in four wells to be used as blank. In the bottom wells, 750 µL of DMEM/F-12 (1:1) media with 10% fetal bovine serum was added. Plates were read at 2, 4, 24, and 48 h after plating the cells using Cytation 3 Multi-Mode Reader (BioTek Instruments, Inc., VT, USA) with top and bottom reading ability, at 492 nm excitation and 517 nm emission wavelengths. The invasion index was calculated based on relative fluorescent units (RFU) using the formula: (RFU of cells at the bottom/RFU of cells at top + RFU of cells at bottom) × 100.
