Matrigel coated filter inserts

Matrigel-coated filter inserts (8 μm pore size) that fit into 24-well invasion chambers were obtained from Becton-Dickinson. Breast cancer cells to be tested for invasion were resuspended in culture medium (5 × 10⁵ cells/well) and then added to the matrigel-coated upper compartment of the invasion chamber in the presence or absence of 20 μM ZER. Fresh culture media with 5% FBS were added to the lower compartment of the invasion chamber. The chambers were incubated at 37°C for 24 h. After incubation, the cells on the upper side of the filter were removed using cotton swabs, and the bottom filters were fixed in 100% methanol, washed in 1× PBS, and stained using toluidine blue dye. Breast cancer cells that invaded through the matrigel were located on the underside of the filter. These cells were photographed using a Scanscope XT apparatus (Aperio Technologies Inc., CA, USA). The invasion rates were calculated by averaging the total number of cells from four filters.

As in a previous study [9 (link)], 4T1 and MDA-MB231 breast cancer cells (2 × 10⁵ cells/well) were resuspended in Matrigel-coated filter inserts (8-μm pore size, Becton-Dickinson, San Diego, CA, USA) and added to the upper compartment of the insert chamber in the presence or absence of 20 ng/mL mIL1A, IL1A, or 30 μg/mL ETL. Fresh culture media (700 μL) was added to the lower compartment of the insert chamber. The chambers were incubated at 37 °C for 16–24 h. After incubation, the upper compartment of the insert chamber was removed using cotton swabs and the bottom filters were fixed and stained (toluidine blue, Sigma-Aldrich, St. Louis, MO, USA). The breast cancer cells that invaded through the Matrigel were located on the underside of the filter. The cells on the underside of the filter were photographed using a CK40 inverted microscope (Olympus, Tokyo, Japan).