Digital sight ds u1 microscope

The upper side of the Transwell insert (6.5-mm diameter insert, 8-µm pore size; Corning Incorporated, Corning, NY, USA) was coated with 50 µl 1 mg/ml Matrigel^® basement membrane matrix (10.4 mg/ml; BD Biosciences) diluted in EBM-2. Aliquots (100 µl) of HUVECs (5×10⁴ cells/ml) resuspended in EBM-2 were added to the upper compartment of the Matrigel-coated Transwell and 600 µl EBM-2 was added to the lower compartment. Following serum starvation with EBM-2 for 2 h, cells were incubated for 15 h at 37°C in EGM-2 Bullet kit media in the presence or absence of ETR (1–25 µg/ml). The inserts were fixed with 95–100% methanol (Merck KGaA, Darmstadt, Germany; #106009.1011) and the non-invasive cells were removed from the top of the membrane using a cotton-tipped swab. Following staining with 0.04% Giemsa solution, the number of invasive cells was determined from six fields using ×200 objective magnification. Images were captured using a Nikon Digital Sight DS-U1 microscope (Nikon Corporation).

Cell migration was quantified via in vitro wound-healing assay as described previously (24 (link)). Following plating of cells on 48-well plates (4×10⁴ cells/well) and allowing them to grow to confluence, a single wound was created in the center of the cell monolayer by gentle removal of the attached cells using a sterile plastic pipette tip. Following serum starvation with EBM-2 for 2 h at 37°C, cells were incubated for 16 h at 37°C in EGM-2 BulletKit medium in the presence or absence of ETR (1–25 µg/ml). Cells were fixed with methanol and then stained with 0.04% Giemsa solution (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Migration of the cells into the wound was observed, and still images were captured following incubation for 16 h. Images were captured using a Nikon Digital Sight DS-U1 microscope (Nikon Corporation, Tokyo, Japan).