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The endothelium-derived Ea.hy926 cell line expresses several inflammation and neoangiogenesis markers in response to activation factors (TNF and VEGFA). The aim of the current study was to compare the expression of the known endothelial cellular markers including VEGFR2 and αvβ3-integrin and other on 2D and 3D cultures (spheroids) of Ea.hy926. According to confocal microscopy, flow cytometry and RT-PCR data the expression of αvβ3-integrin (green), ICAM-1(red), E-selectin (blue) increased on the surface of TNF-activated Ea.hy926 cells grown in 3D culture in comparison with 2D. Thus, 3D cultures of the Ea.hy926 cell line represent a promising model for the inflammation studies.