24 well transwell unit with polyethylene terephthalate membranes

Migration studies were performed as previously described [20] (link). Briefly, using a 24-well transwell unit with polyethylene terephthalate membranes showing a pore size of 8.0 µm (BD Falcon, Basel, Switzerland). MCF-7 (3.10 4 ) or MDA-MB-231 (5.10 3 ) cells in serum-free RPMI were loaded into the upper compartment. The lower chamber contained treatments in 10% FBS/RPMI medium. After 20 h (MDA-MB-231) or 40 h (MCF-7) at 37ºC non-migrated cells on the upper surface of membranes were gently scrubbed with a cotton swab. Migrated cells were fixed in 4% formaldehyde/PBS and stained with 0.5% crystal violet. Total migrated cells were counted with an Olympus Bx50y microscope and photos were taken with an Olympus DP73 camera. Results were expressed as the number of migrated cells compared to control. For invasion assays, transwell units were previously coated with Matrigel® (BD Biosciences, MA, USA)/RPMI (1:2) and MCF-7 (1.10 5 ) or MDA-MB-231 (1.10 4 ) cells were loaded in the upper chamber. Cells that invaded through Matrigel were fixed, stained and counted as previously described for migration studies.

Cell migration was investigated using a 24-well transwell unit with polyethylene terephthalate membranes showing a pore size of 8.0 m (BD Falcon, Basel, Switzerland). Equal number of MCF-7 or MDA-MB-231 cells in serum-free RPMI was loaded into the upper compartment. The lower chamber contained RPMI medium plus 10% FBS. After 20 h at 37 • C non-migrated cells on the upper surface of membranes were gently scrubbed with a cotton swab. Cells migrated were fixed in 4% formalin and stained with 0.5% crystal violet. Total number of migrated cells was counted. For the invasion assay, transwell units were coated with Matrigel ® (BD Biosciences, MA, USA)/RPMI (1:2). After 20 h at 37 • C, cells that invaded through Matrigel and reached to the reverse side were fixed, stained and counted under a microscope. Controls to verify that cell proliferation was not affecting migration/invasion results after 20 h incubation were performed (data not shown).