Matrigel basement membrane like matrix

A Matrigel transwell invasion assay was performed to examine the effect of NF-κB/p65 inhibition on the cell invasion process. Approximately 10⁴ cells were seeded in medium containing 1% FBS in the presence or absence of DHMEQ (10 μg/ml) onto the upper region of the transwell chamber (8-μm pore size; Corning) coated with a Matrigel basement membrane-like matrix (1 mg/ml; BD). The lower chamber of the transwell was filled with medium containing 10% FBS. After a 24-h incubation at 37°C and 5% CO₂, the cells were fixed with absolute ethanol and stained with crystal violet, and the non-invaded cells present in the inserts were removed with a cotton swab. Images of the invaded cells were acquired using the same equipment described above. Five random fields were photographed (x200 magnification) and counted. The values of non-treated cells were defined as 100%, and the relative proportion was calculated for DHMEQ-treated cells. The experiments were performed in triplicate.

The Matrigel transwell invasion assay was conducted to evaluate the effect of APX2009 treatment on the cellular invasion process. Approximately 30,000 and 100,000 cells of MDA-MB-231 and MCF-7, respectively, were seeded onto a medium containing 1% FBS with APX2009 (0.8 or 4 μM for MDA-MB-231 and 4 or 20 μM for MCF-7, non-lethal concentrations) or vehicle (DMSO) in the upper side of the transwell chamber (8 μm pore size; Corning, USA) that was coated with a Matrigel basement membrane-like matrix (1 mg/mL; BD). The lower chamber of the transwell was filled with a medium containing 10% FBS. After 24 h of incubation at 37°C and 5% CO₂, the cells were fixed with absolute ethanol and stained with crystal violet (0.5% in 20% ethanol). The non-invaded cells present in the inserts were removed with a cotton swab. Images of the invaded cells were acquired, and five random fields were photographed (100× magnification) and counted using ImageJ software (v.1.8.0_112).