Trans well migration (Boyden chamber) assay

The apical chamber of 24-well cell culture inserts (Millipore) were seeded with equivalent numbers (250,000) of previously starved cells of interest in serum-free medium as previously described [6, 9]. Culture medium containing 30% FBS and 5 μg/ml fibronectin was added to the basal chamber of the unit. Cells were allowed to migrate through the membranous barrier for 22 hours at 37°C. Following incubation, cells that did not migrate were removed from the apical side of the filter by wiping with PBS-wetted Kim wipe, followed by PBS rinse to remove remaining cell debris. The filters containing migrated cells were fixed with 4% paraformaldehyde (PFA) for 5 minutes, washed twice with 1x PBS, permeabilized with 100% methanol for 25 minutes, labeled with Giemsa stain (for 15 minutes at room temperature), and imaged using a Nikon Eclipse Ti microscope adjusted with a Nikon digital sight camera. Images were processed using Image J software. Cells from 3 to 5 different fields were blind-counted and averaged. In parallel, cells that migrated to the basal chamber were enumerated using the hemocytometer cell counting method. Number of migrated cells was calculated as:

In parallel, basal chamber cells were collected, number enumerated and presented as cells in basal chamber ×10³. In addition, basal chamber cells were seeded in a 96 well plate for 5 hours, medium removed and cells stained with 0.5% crystal violet for 30 min. After washing with 1X PBS twice, cells were treated with 0.2 % SDS for 10–15 min. Absorbance was read with a Tecan microplate reader at 550 nm. Spectrophotometric absorbance correlates with cell migration, where increased absorbance correlates with higher cell motility [49].