8.0 μm cell culture inserts

Transwell migration assay was performed by using 8.0-μm cell culture inserts (BD Biosciences) to test the migratory ability of primary breast cancer cells, MCF-7/T47D cells, and mammosphere-forming cells. Cells were seeded at 2.5 × 10⁵ cells per well in serum-free DMEM in the upper chamber of 12-well plates and allowed to migrate for 8 hours toward DMEM containing 10% FBS in the lower chamber. After 8 hours, the cells in the upper chamber were removed with a cotton swab and the migrated cells in the lower surface of the membrane were fixed and stained with giemsa or the migrated fraction of 2° mammospheres were collected from the under-surface of the membranes after 24-hour migration assay for flow cytometry. Images were acquired with a brightfield microscope (Leica, Wetzlar, Germany) at 20× magnification. To quantify migratory cells, three independent fields were analyzed by using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Migration was expressed as percentage of cells migrated. For the same, the percentage of cells that migrated in the control set of each relevant experiment was taken as 100%.

Migration was assessed using a Boyden chamber assay. ECs (20,000) were seeded in 8.0 μm cell culture inserts (BD Biosciences) in serum-free media, and placed into 24-well plates containing serum-free media, or CM. For inhibitors, SB505124 [2 μM], ruxolitinib [100 nM] or DMSO [0.05%] were added to the inserts and wells. After 16 h, cells were fixed in methanol and stained. The total number of cells that migrated was counted.