Transwell membrane insert

ACSs were seeded into 60 mm plates and treated with udenafil, IL-4, or IL12B for 3 days. ASCs starved for 1 day (1.5×10⁴ cells/well) were suspended in a serum-free medium and seeded on the upper side of transwell membrane insert (BD Falcon, CA, USA), which was pre-coated with matrigel (1/60 dilution, BD Matrigel matrix, CA, USA). The normal serum with FBS was added in lower plate as chemoattractant. The cultures were incubated for 1 day to allow transwell migration. The inserts were then removed, and their upper surface was cleaned using cotton swabs and washed with PBS to remove non-migrating cells. The inserts were stained with 0.1% formalin/10% crystal-violet solution (Sigma-Aldrich) for 20 min, and cell number was analyzed under a ZEISS Observer.D1 microscope. Multiple images (15–20) were acquired per insert, and the average cell counts were calculated.

A total of 200 μL of a cell suspension containing 5×10⁴ cells in FBS-free DMEM was seeded in the upper chamber of Transwell membrane inserts with 8 µm pore size (BD Biosciences). Approximately 500 μL of DMEM supplemented with 10% of FBS was placed into the lower chambers. The filter membranes precoated with Matrigel (BD Biosciences) were employed for the in vitro invasion assay, whereas the in vitro migration assay was carried out with filter membranes not coated with Matrigel. After 24 h incubation, a cotton swab was used to gently remove the cells remaining on the upper side of the membrane surface. The cells that passed through membranes were fixed with 4% formaldehyde and stained with a 0.5% crystal violet solution. The numbers of migrated and invaded cells were determined in five randomly chosen visual fields under an Olympus inverted light microscope (×200 magnification; Olympus Corporation, Tokyo, Japan).