2.12. Transwell Migration Assay

Migration of TNBC cells: TNBC cells were continuously treated by TNFα + IL-1β/vehicle. Migration assay of TNBC cells towards 10% FBS were for 11 h using transwell inserts with 8 μm pores (#3422, Corning, Corning, NY, USA) that were precoated with fibronectin (20 µg/mL, diluted in serum-free DMEM; #03-090-1; Biological Industries) for 1 h at 37 °C.

Migration of THP-1 cells: MDA-MB-231 cells that were continuously stimulated by TNFα + IL-1β were cultured overnight in complete media supplemented with TNFα + IL-1β. Then, the media were replaced with fresh DMEM containing 0.5% FBS and were supplemented with the glycolysis inhibitor (2-DG) (concentration as in ELISA assays), without TNFα + IL-1β. Control cells were supplemented with the inhibitor solubilizer. After 2 h, TNFα + IL-1β were added without a wash. After 24 h, the media were washed and replaced by fresh TNFα + IL-1β-deprived and inhibitor-free DMEM, which did not contain FBS. After 24 h the CM were collected, filtered through 0.45 μm membrane to discard of cells and concentrated 2×. Then, migration assay of THP-1 cells towards the CM were performed for 75–90 min using transwell inserts with 5 μm pores (#3421, Corning).

In both analyses, the migrated cells were fixed and stained using Hemacolor (#111661, Merck), were photographed and counted at multiple fields.