Falcon transwells

MDA‐MB‐231 (3 × 10⁵) with ME1 knockdown or a scrambled control were placed in a DMEM medium supplemented with 2% inactivated FBS (Invitrogen, Carlsbad, CA, USA). These cells were then seeded onto the upper chamber of Falcon transwells (Falcon, Corning, USA) for migration assay. THP‐1 cells were induced macrophage by PAM, then THP‐1‐induced macrophage cells (8 × 10⁴) were seeded in the lower chamber containing RPMI 1640 growth medium as described above. Subsequently, the cells were placed in a CO₂ incubator at 37°C for 7 h. After the incubation period, any remaining cells in the upper chamber were removed using cotton swabs, whereas cells on the undersurface of the transwells were fixed using a 10% formaldehyde solution. The cells were then stained with crystal violet solution, and the number of breast cancer cells was determined by counting three fields with a phase‐contrast microscope. Each experiment was completed three times to ensure accuracy.

For macrophage cell infiltration, THP‐1 cells were induced macrophage by PAM, then THP‐1‐induced macrophage cells (5 × 10⁵) were seeded in the upper chamber of Falcon transwells (Falcon, Corning, USA) containing RPMI 1640 medium without FBS. MDA‐MB‐231 cells (8 × 10⁴) with ME1 knockdown or a scrambled control were placed in lower chamber containing DMEM medium supplemented with 10% FBS. Subsequently, the cells were placed in a CO₂ incubator at 37°C for 24 h. After the incubation period, any remaining cells in the upper chamber were removed using cotton swabs, whereas cells on the undersurface of the transwells were fixed using a 10% formaldehyde solution. The cells were then stained with crystal violet solution, and the number of macrophage cells was determined by counting three fields with a phase‐contrast microscope. Each experiment was completed three times to ensure accuracy.

The invasion ability of breast cancer cells was assessed in vitro by employing a transwell assay, as described in our previous study.
²⁹ (link) In summary, total of 3 × 10⁵ breast cancer cells (Hs578T or MDA‐MB‐231) with ME1 knockdown or a scrambled control were placed in a suspension containing 2% FBS. These cells were then seeded onto the upper chamber of Falcon transwells (Falcon, Corning, USA), which were coated with Matrigel (BD Biosciences, MA, USA) to facilitate the invasion assay. Subsequently, the cells were placed in a CO₂ incubator at 37°C for either 12 or 24 h. After the incubation period, any remaining cells in the upper chamber were removed using cotton swabs, whereas cells on the undersurface of the transwells were fixed using a 10% formaldehyde solution. The cells were then stained with crystal violet solution, and the number of breast cancer cells was determined by counting the three fields with a phase‐contrast microscope. Each experiment was completed three times to ensure accuracy.