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Leukocyte rolling on the blood vessel wall represents the first step in the process of inflammation. In this study, nanofabricated substrates were designed with two different sets of feature size and spacing to mimic the expected distribution of discrete molecular adhesion patches on the surfaces of endothelial cells lining the blood vessel wall. P-selectin was attached to these nanopatterned dots, and the rolling behaviour of HL60 cells was analysed as a function of wall shear stress. When wall shear stress was less than 1 dyne/cm2, rolling velocity was independent of substrate patterning. However, when wall shear stress was higher than 2 dyne/cm2, rolling velocity was increased on the patterned substrates compared with the unpatterned sample, and rolling velocity increased with nanodot spacing distance. The influence of pattern spacing on the waiting time, the duration of zero-velocity pauses during rolling, also increased for wall shear stresses greater than 2 dyne/cm2. Additionally, the variance of instantaneous rolling velocities increased among substrates when the shear stress was greater than 6 dyne/cm2, indicating that the spatial arrangement of the nanodot pattern influenced not only the average velocity with which the cells rolled but also the saltatory nature of rolling. These results suggest that nanodot substrates represent a tool to investigate the biophysical and biochemical mechanisms regulating dynamic adhesion of leukocytes to the blood vessel wall.