Permeable support for 24 well plate with 8.0 μm transparent pet membrane

The D492HER2 cells were cultured with siRNA transfection (Scramble and siGFPT2) for 48 h in a 6-well plate. GFPT2 knockdown followed the methods described above. Cells were then reseeded into filter units (Falcon Permeable Support for 24-well Plate with 8.0 μm Transparent PET Membrane, 353097) coated with Matrigel (Corning Matrigel Matrix, 356234) at a density of 30,000 cells/well. First, the filter inserts were coated with 100 μl 1:10 diluted Matrigel for 20 to 30 min at 37 °C. Next, 300 μl of cell suspension was added on top of the filter units. Then, 500 μl of H14 medium with 10% FBS was added to the wells in the 24-well plates below the filters. Finally, cells were incubated at 37 °C and 5% CO₂ for 48 h. Noninvasive cells on top of the filters were removed with cotton swabs, followed by fixation with paraformaldehyde (PFA, 3.7%, Sigma, 252549) and DAPI staining (1:5000, Sigma, D9542). Ten images per filter unit were taken by the EVOS FL Auto Imaging System (10×, Thermo), followed by the batch analysis of the images in Macro ImageJ 1.52p. To normalize the different cell numbers in the filter units, cells were seeded into a 24-well plate along with the filter units, and they were cultured and treated in the same way as cells in the filter units.