FIG. 4.
Effect of microfluidic patterning time on mP-selectin deposition and neutrophil rolling. Immunofluorescence images of glass coverslips coated with mP-selectin utilizing μFP perfusion times of (a) 5 min, (b) 10 min, or (c) 15 min. The protein line patterns were formed by perfusing a 0.5 μg/ml mP-selectin solution through microfluidic channels (300 μm × 50 μm cross-section) at a perfusion shear stress of 16.0 dyn/cm2. (d) Representative fluorescence intensity line profiles for the different μFP perfusion times. (e) Mean neutrophil rolling velocities on P-selectin coated substrates fabricated with μFP patterning times of 5, 10, and 15 min. The protein patterns for the rolling experiments were formed by perfusing a 0.1 μg/ml mP-selectin solution through microfluidic channels (1000 μm × 50 μm cross-section) at a perfusion shear stress of 16.0 dyn/cm2. No rolling was observed for static patterns at 15 min of patterning time. Data are shown as the average value ± SEM and were derived from three to four experiments with blood from at least three different donors. The number of cells analyzed for each point ranged from 32 to 104 cells. Statistical significance was assessed by comparing each concentration curve to the 15 min curve using a two-way ANOVA with a post-hoc Sidak's multiple comparison test: * P < 0.05 and *** P < 0.0001.