An in vitro neutrophil transmigration assay was used to investigate the effects of HUVEC substrate stiffness on neutrophil transmigration. (A) HUVECs were plated onto fibronectin-coated polyacrylamide gels of various stiffnesses from 0.42 kPa to 280 kPa. After monolayer formation, HUVECs were treated with TNF-α to induce an inflammatory response. Neutrophils were isolated from human blood and plated onto the HUVEC monolayer. (B) The fraction of neutrophils that transmigrated (as described in “Transmigration assays”) was quantified as a function of the stiffness below the HUVECs. Bars represent average fraction of transmigrated cells. Error bars represent SE of 3 to 8 experiments (N = 6, 8, 4, 6, 8, 3, 7, and 3 from 0.42, 0.87, 3, 4, 5, 13, and 280 kPa, and glass [∼ 50 GPa], respectively). (C) Data from panel B, up to 5 kPa, are plotted with a linear fit (r² = 0.99). (B-C) ***P < .001, **P < .005, or ^∧P < .05, using a t test compared with 5 kPa value of same cell type. (D) Shown is an example of a phase-contrast time lapse image sequence of 4 neutrophils, 2 of which transmigrate through the endothelium. Scale bar represents 10 μm and applies to all images. T = 0 is time just before initiation of transmigration in the first cell. White arrows point to the phase-darkened portion of the neutrophil as it transmigrates through the endothelium. At T = 75 seconds, the white arrowhead points to a neutrophil that possibly initiates but does not complete transmigration. In the final frame, the 2 darkened neutrophils are between the endothelium and the gel, whereas the 2 white neutrophils are on top of the endothelium.