Gfr matrigel matrix

HUVECs were seeded on 6-well plates and transfected with miR-26a mimics using RNAimax (Invitrogen). After 24 h, cells were trypsinized, counted, and seeded at 1.5 × 10⁴ cells/well in 48-well plates pre-coated with phenol-red free, growth factor reduced (GFR) Matrigel Matrix (BD Bioscience). For treatments of VEGF (R&D Systems, Minneapolis, MN, USA), it was suspended in EBM-2 medium and used at the concentration of 50 ng/ml. The formation of tube-like structures was observed every 2 h under an optical microscope at 40× magnification and quantified with the Image J software (Open Access, Public Domain).

For transwell migration assays, polycarbonate cell culture inserts with 8-μm pore size and 0.47 cm² growth area (ThermoFischer) were used. The inserts were coated with 50 μl growth factor reduced (GFR)-Matrigel™ matrix (BD Biosciences) diluted 10 times in serum-free media and incubated at 37°C for 1.5 h. A total of 200 μl serum-free media containing 5 × 10⁴ serum-starved HK-2 cells were placed in the top well chamber, whereas the bottom chamber was filled with 500 μl complete medium enriched with up to 10% FBS. Media in the top and bottom chambers were supplemented with vehicle or TGFβ. After 24 h of treatment, cells were washed in PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100. Cell nuclei were then stained with Hoechst (ThermoFischer). Cells from the top surface of the membrane were gently removed with a cotton swab. Membranes were then cut out from the inserts with a scalpel blade and mounted on microscope slides using antifade mountant (ThermoFischer), with the lower surface facing up. Nuclei were visualized with an Olympus IX71 fluorescence microscope and counted using Image J.