Boyden Chamber Assay for Cell Migration

A Boyden chamber assay was used, as described previously (17, 29 (link)). Briefly, cells were trypsinized, suspended in 0.1% BSA/RPMI, and seeded (2.5 × 10⁴ cells/well) in the upper compartment of a Boyden chamber (NeuroProbe). A 12-μm-pore Matrigel-coated polycarbonate membrane was used to separate the upper and lower compartments. In the lower chamber, RPMI medium containing 10% FBS was used. After an incubation period of 16 hours at 37°C, membranes were recovered and cells on the upper side of the membrane (nonmigrating) were wiped off the surface. Migrating cells on the lower side of the membrane were fixed and stained with the Hema 3 Staining kit (Thermo Fisher Scientific). Migrating cells in each well were counted in five random fields by contrast microscopy using an Eclipse E200 Nikon microscopy (4X magnification) and the ImageJ/Fiji software.