Transwell chamber with a polycarbonic membrane

The cell migration used a transwell chamber with a polycarbonic membrane (6.5 mm diameter and 8 μm pore size; Corning). The serum-free DMEM of no-transfected or transfected cell was added to the upper chamber, and 600 μl of 15% FBS–DMEM was added to the lower chamber. The cells that were migrated to the lower chamber surface were fixed with 4% methanol and stained with 10% Giemsa (Solarbio, China), and the unmigrated cells on the membrane were wiped off with cotton swabs.

Human CFs migration in vitro was assayed using a transwell chamber with a polycarbonic membrane (6.5 mm diameter and 8 µm pore size; Corning). Human CFs were trypsinized and re‐suspended in serum‐free medium at a density of 5 × 10⁴ cells/mL. Two hundred microlitre of the no‐transfected or transfected cell suspension was added to the upper chamber, and 600 µL of serum‐free DMEM/F12 supplemented with different concentrations of TGFβ3 (0, 5, 10 or 50 ng/mL) or Ang II of 100 nmol/L was added to the lower chamber according to the experimental requirements. Non‐migrating cells on the top surface of the membrane were removed with cotton swabs. The cells that migrated to the lower surface of the membrane were fixed with 4% methanol and stained with 10% Giemsa (Solarbio). The cell number was measured within five randomly chosen fields at 200 × magnification, and the average number was calculated with ImageJ.