PMC

Changes in the intrinsic rate of bond formation with increasing contact stress. Open bars indicate results obtained with tosyl-activated beads and shaded bars indicate results obtained with protein-G-conjugated beads. Site densities on the beads were ∼140 sites/μm² for 2.8-μm beads, ∼290 sites/μm² for 4.5-μm beads, ∼640 sites/μm² for 3-μm beads, and ∼800 sites/μm² for 10-μm beads. Contact areas were in the range of 6.0 μm² for all measurements. The expected bond number was calculated according to . Contact stress was calculated according to . Increasing contact stress results in a significant increase in the expected bond number.

This series of video micrographs shows a neutrophil in contact with an ICAM-1-coated bead (4.5-μm diameter) at four different impingement pressures. Beads were held approximately one neutrophil diameter within the translation pipette to maintain alignment of the cell and bead. The impingement forces for the different images (from A–D) were ∼70 pN, ∼140 pN, ∼210 pN, and ∼280 pN. The contact area between the two surfaces was determined from measurement of the contact length L_con, the distance between the two points in the cross-section where the bead and cell intersect. (See vertical line in D.)

Schematic of the force balance on an adherent spherical cell under shear flow. The fluid exerts a drag F_s and a torque T_s on the sphere, creating a moment on the cell that is balanced by the reaction (peeling) force at the trailing edge of the cell F_p and a contact stress σ between the cell and the substrate. The cell radius is R_s and the radius of the contact zone is R_c. The drag and torque on the cell were estimated from the results of and the peeling force was obtained by taking a moment balance around the center of the contact zone. The contact stress must balance the vertical reaction force of peeling, F_p, and is assumed to be distributed uniformly over the entire contact area

Relationship between the expected bond number, 〈n〉, and contact area. The values of 〈n〉 were corrected to account for nonspecific adhesion. (A) Data is shown for all cells tested, each cell represented by a different symbol. Solid lines show the linear regressions to each cell as a guide for identifying different points from the same cell. For most of the cells tested, the Pearson's r-value for the fits ranged from 0.8 to 0.98, but two cells have values of 0.54 and 0.58 and one cell had a value of 0.73. (B) The same data grouped according to impingement pressure. Error bars represent standard deviations of the distribution of force (horizontal bars) and expected bond number (vertical bars). The solid line is a linear regression to the four mean values weighted by the inverse of the variance (Pearson's r = 0.99). The dashed line is a linear regression to the data at the highest three pressures, fixed at the origin (Pearson's r = 1.0).

Adhesion probability of neutrophils binding to ICAM-1 beads as a function of the impingement force. (A) Increase in the adhesion probability with force in the presence of 5 mM Mg²⁺ for three individual cell-bead pairs. Different symbols correspond to different cells. Pearson's r-coefficients for the fits were 0.96, 0.97, and 0.98. (B) Mean adhesion probability for 12 cell-bead pairs as a function of impingement force. Data are grouped by impingement pressure and error bars represent the standard deviation of the values obtained for the 12 cells tested (▪). Addition of an IgG control antibody caused a statistically insignificant increase in the adhesion probability (⋄). Nonspecific adhesion probability under control conditions (see C) is indicated by the solid line with 95% confidence intervals shown by the dotted lines. (C) Several conditions were used to determine the level of nonspecific binding and demonstrate specificity of β₂-integrin binding to the ICAM-1 beads. The conditions were 5.0 mM Mg²⁺ plus 10 μg/mL of the β₂-blocking antibody IB4 (□), 5.0 mM Mg²⁺ with NCAM-coated beads (★), 1.5 mM Ca²⁺ (•), and 1.5 mM Ca²⁺ plus 10 μg/mL IB4 (○). Data is reported as the mean ± SD and grouped by the impingement pressure. The lines indicate a variance-weighted linear regression to the mean values, and dashed lines show the 95% confidence interval of the fit. Note the different vertical scale in C.