Inform software ver 2

A cell migration assay was carried out using the Boyden chamber technique, as previously reported^{35 (link)}. Cell culture inserts (8-μm pore size, 6-mm in diameter) were used according to manufacturer’s instructions. Invasion assays were performed using Corning Matrigel invasion chambers (pore size: 8 μm; Discovery Labware Inc., Woburn, MA, USA) as previously reported^{21 (link)}. Cells were plated at a density of 1 × 10⁵ cells/500 μL on the upper surface of the inserts, and 4 h later for migration and 16 h later for invasion, cells that had migrated through the membrane to the lower surface of the filter were fixed and stained with a Diff-Quik staining kit (Polysciences, Inc., Warrington, PA, USA). The images were taken using a Mantra multi-spectral microscope (Perkin-Elmer), and then the images were loaded into inForm software ver. 2.4 (Perkin-Elmer) to count the number of migrated or invasive cells in 12 random fields under a 20 × magnification objective. All independent experiments were repeated twice in triplicates.

Cell culture inserts (8- µm pore size and 6-mm diameter; Corning, Glendale, AZ, United States) were used according to the manufacturer’s instructions. HASMC growth media was placed in the lower chamber of a 24-well plate. Cells were plated at a density of 1 × 10⁵ cells/500 µL SmGM™-2 without supplements on the upper surface of the inserts. Following 6 h of incubation at 37°C, cells that had migrated through the membrane to the lower surface of the filter were fixed, stained with a Diff-Quik staining kit (Polysciences, Inc, Warrington, PA, United States), and then imaged using Mantra, multi-spectral microscopy. The images were loaded into the inForm software ver. 2.4 (Perkin-Elmer, Inc, Waltham, MA, United States) to establish the number of migrated or invaded cells in 12 random fields at ×20 magnification.