Transwell invasion and migration assay

Millipore Transwell chambers (pore size: 8 μm) was used to perform Matrigel invasion assay. We combined 500 μL of medium containing 15% FBS with 100 μL and added the mixture into the lower chamber of a 12-well plate to induce cell migration ⁴³. Cells were treated with PBS (control), GM (1 g/L GMs), PRP (1 g/L PRP), and GM+PRP (4 g/L GM+PRP). We added 2×10⁴ keratinocytes coated with growth-factor reduced Matrigel (Corning), and serum-free medium (500 μL) into the upper chamber of the plate. The plate was incubated for 24 h at 37^◦C. The upper surface of the membrane of cells was cleaned up using a cotton swab before incubation was over. After migration, the cells were treated with methanol (Merck) through the lower surface of the membrane and stained with Giemsa (HiMedia). The images of cells were obtained using an inverted microscope (CKX41, Olympus; Shinjuku, Tokyo, Japan). NIH ImageJ software (dVersion 1.46, Bethesda, MA, USA) was used to count the cells in five fields-of-view. The experiment was conducted in triplicate.