Transwell cell migration assay

Boyden chamber assay experiments were conducted using a 24-well Transwell system (6.5 mm Transwell (#3422), Corning) with each well separated by a microporous 10 μm thin transparent polycarbonate membrane (8 μm pore size) into an upper(‘insert’) and a lower chamber (‘well’). A volume of 100 uL containing 40,000 cells were plated to each insert and 600 uL medium was added to the wells. Cells in ‘insert’ were allowed to migrate for 24 h. Then, cells were fixed and stained in a 20% methanol/0.1% crystal violet solution during 1 h at 4°C temperature, followed by washing ‘insert’ with water to remove redundant staining. Cells on upper part of ‘insert’ membrane were wiped out. Cells migrated onto lower part of ‘insert’ membrane were photographed, counted and presented as bar graph.