Matrigel coated membrane 24 well insert

For invasion assays, 2 × 10⁴ cells were plated in the top chamber with a Matrigel-coated membrane (24-well insert, BD Biosciences, NJ, USA). Cancer cells were plated in RPMI1640 medium without FBS and RPMI1640 medium supplemented with 10% FBS (for all other cells) was used as a chemoattractant in the lower chamber. The cells were incubated for 24 h, and cells that did not migrate or invade through the pores were removed using a cotton swab. Cells on the lower surface of the membrane were stained with the Diff-Quick Staining Set (Sysmex, Hyogo, Japan) and counted. All assays were performed in triplicate. The data are expressed as the invasion percentage through the Matrigel matrix and membrane relative to the migration through the control membrane, according to the manufacturer’s instructions.

The experiment was performed with 1.5 × 10⁵ cells and Matrigel‐coated membrane (24‐well insert, BD Biosciences; pore size, 8 μm). Migrated cells on the lower surface of the membrane were stained with crystal violet, and five randomly selected fields were counted.

For transwell migration assays, 2.5 ~ 5 × 10⁴ non-adherent BC cell line MDA-MB-453 cells were plated in the top chamber with the non-coated membrane (24-well insert; pore size, 8 μm; BD Biosciences). For invasion assays, 1.25 × 10⁵ cells were plated in the top chamber with Matrigel-coated membrane (24-well insert; pore size, 8 μm; BD Biosciences). In both assays, cells were plated in medium without serum, and medium supplemented with 480 ng/mL recombinant C5a were used as a chemoattractant in the lower chamber. Cells were incubated for 12 h and cells that did not migrate or invade through the pores were removed by a cotton swab. Cells on the lower surface of the membrane were stained with the crystal violet dye and counted. The results were observed under the microscope. Magnification: 200 ×.