Fig 1: The instantaneous rolling velocity of beads by interaction of integrin αvβ3-fibronectin (FN)/truncated fibronectin (FN1.3). (A) Shown are instantaneous velocities of five representative free flowing microspheres under various shear stress of 0.1 (black line), 0.2 (red line), 0.3 (green line), 0.5 (dark blue line) and 0.7 dyn/cm2 (wathet line). (B-F, H-L) Representative instantaneous velocities of FN (B–F)/FN1.3 (H–L) bearing microspheres of 3 μm radius flowing over and being arrested on a surface coated with integrin αvβ3 (200 ng/mL) at wall shear stress below, equal and above the shear optimum (0.1, 0.2, 0.3, 0.5 and 0.7 dyn/cm2). The data were recorded at 100 fps. The instantaneous velocities were set as zero when their values were below 50 μm/s when the shear stress stronger than 0.2 dyn/cm2, while instantaneous velocities were set as zero when their values were 15 and 25 μm/s at 0.1 dyn/cm2 and 0.2 dyn/cm2, respectively. (G) Free flowing beads (pentagon) were referred to the sliding beads on the same focal plane, which has not any interaction with other molecular. The R value of the fitted curve is 0.9946.
Fig 2: Measurement of transient tether lifetime and dissociation rate constant, k off . Lifetimes of transient tethers of fibronectin (FN) and truncated fibronectin (FN1.3)-bearing beads (A, E) on a surface with low-density integrin αvβ3 were plotted against wall shear stress. The dissociation rate constant, k off (B, F), derived from negative slope by linear fitting the tether lifetime plot of ln (number of events with a lifetime ≥ t) vs. t for low shear stress 0.1–0.3 dyn/cm2 (C, G) and high shear stress 0.3–0.7 dyn/cm2 (D, H). Data represent the mean ± SD of three experiments.
Fig 3: Schematic of the parallel plate flow chamber and specific experiment. (A) The flow chamber was assembled by a lexan, a gasket (length × width × thick = 2 × 0.5 × 0.0254 cm3) and 35 mm dish. There were three channels on the Lexan, including inlet, outlet, and vacuum. (B) The distance-time curve tracked by Image Plus Pro (IPP) software. Lifetime was calculated according to the upper graph from t1 to t2 (C) Adhesion rate of fibronectin (FN)/truncated fibronectin 1.3 (FN1.3) functionalized microspheres on phosphate-buffered saline (PBS) adn bovine serum albumin (BSA)-blocked, and integrin αvβ3-immobiled substrates. Data represent the mean ± SD of three experiments. The significance of the difference is shown by p-value, with ns. for p > 0.05, and ** for p < 0.005. (D, E) Instantaneous velocity and cumulative distance of one tethering event. (F, G) Instantaneous velocity and cumulative distance of one rolling event.
Fig 4: Rolling stop time and stop frequency of beads by interaction of integrin αvβ3 with fibronectin (FN) or truncated fibronectin (FN1.3). (A, B) Mean stop times and (C, D) stop frequencies for FN/FN1.3-bearing microspheres rolling on substrate coated with integrin αvβ3 (200 ng/mL) were plotted against wall shear stress. The data were recorded at 100 fps, and the mean ± SD of three independent experiments is presented.
Fig 5: Rolling velocity and reduced percent of rolling velocity of beads by interaction of integrin αvβ3 with fibronectin (FN) or truncated fibronectin (FN1.3). (A, B) Velocity of FN-coated beads (blue square) and FN1.3-coated beads (red square) rolling on integrin αvβ3- immobilized bottom. (C, D) The reduced percent of rolling velocity of FN-coated beads (red diamond) and FN1.3-coated beads (blue diamond). The data were recorded at 100 frames per second, and the mean ± SD of three independent experiments was presented.
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