Eclipse te200 microscope

Endothelial capillary tube-like formation was assessed using Matrigel (Corning Life Sciences, Corning, NY, USA) according to the manufacturer's instructions. A basement Matrigel membrane was diluted to a protein concentration of 5 mg/ml using a sterile medium MCDB131, that is, the same medium that we used for endothelial cell culture, and stored at -20 °C. Before the experiment, a sample of Matrigel was thawed (overnight at 4 °C), plated onto 15-well plates (Ibidi, Martinsried, Germany), and incubated at 37 °C for 30-40 min to allow polymerization. Then, endothelial cells in the complete cell culture medium were seeded onto Matrigel-coated plates; HMEC-1: 3, 000 cells/well, HUVECs 4, 000 cells/well. After 6 h (for HMEC-1) or 8 h (for HUVECs), the created structures were stained with calcein-AM (ThermoFisher, Waltham, Massachusetts, USA) for 15 min. Endothelial cell capillary tubes were assessed by fluorescence and phase contrast microscopy (Nikon Eclipse TE200 microscope, Tokyo, Japan) with a Zeiss CCD video camera (AcioCamERc5s, Oberkochen, Germany). The characterization of the created structures was performed by measuring the number, length, and width of the capillary tubes using the Angiogenesis Analyzer tool in ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej. nih.gov/ij/) [29] .

Cells were seeded in 24-well plates at a density of 400, 000 cells/well. After overnight incubation, cells were pretreated with PAD inhibitors for 16 h before scratching the confluent monolayer. To exclude the influence of endothelial cells proliferation on wound closure, the medium was supplemented with the cytostatic agent mitomycin C (at a final concentration of 10 µg/ml; Sigma-Aldrich, Saint Louis, MO, USA) throughout the experiment. Next, the cells were incubated in inhibitor-free medium, and after 8 h, the size of the wound was measured. Cell migration was tracked using a phase-contrast microscope image analysis system (Nikon Eclipse TE200 microscope, Tokyo, Japan) with a Zeiss CCD video camera (AcioCamERc5s, Oberkochen, Germany). Wound healing inhibition analysis was performed using the Wound Healing Tool plugin in ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https:// imagej.nih.gov/ij/) [30] .