Falcon cell culture insert transwell

MCF-7 cells were seeded into each well of a 24-well plate at 1×10⁵ cells/200 µl/well in culture medium and incubated for 2 hours before coculture. A suspension of MDA-MB-231 or MCF-7 cells (1×10⁵ cells/200 μl) seeded onto a 3‐μm BD Falcon cell culture insert transwell (BD Biosciences) was then inserted into an MCF-7 cell-seeded 24-well plate. The cells were treated with M protein for 48 h before undergoing further examinations.

A suspension of MDA-MB-231 or MCF-7 cells (1×10⁵ cells/200 μl) in IMDM medium was seeded onto a BD Matrigel Basement Membrane Matrix (BD Biosciences, Franklin Lakes, NJ, USA)‐coated 8‐μm BD Falcon cell culture insert transwell (BD Biosciences). A total of 400 μl of IMDM supplemented with 10% FBS was added to the lower compartments of each chamber. The cells were treated with M protein for 24 h. After removing the cells remaining inside the transwell with a cotton swab, the bottom surface of each transwell membrane were then fixed in 4% paraformaldehyde in two minutes, permeabilized in methanol in 20 minutes. Then, the fixed transwell membranes were dipped in Hematoxylin in 30 seconds and washed in distilled water two times, following by dipping in Eosin in 20 seconds and washing in distilled water two times. The membranes were observed under microscope immediately after staining. Five random pictures were taken for each transwell, and the average number of cells was counted using the ImageJ software program. The invasion rate was calculated as follows: