Immunofluorescent analysis of VCAM-1 receptor molecules expressed on human neutrophils. Cells were labeled for (top left) α₄ (using anti-CD49d antibody P1H4); (top right) α₅ (using anti-CD49e antibody SAM-1); (bottom left) α₉ (using anti-α₉β₁ antibody Y9A2); (bottom right) β₁ (using anti-CD29 antibody B-D15) and their corresponding isotype controls.

Interaction between the bead and the cell during the experiment: (A) initial contact; (B) no adhesion; (C) adhesive event. The adhesive event here resulted in the formation of the membrane strand between the cell and the bead. This occurred after some, but not all the adhesive contacts. Original magnification 200× for all panels. (Bar = 5 μm.)

Time and temperature dependent adhesion probability for VCAM-1 coated bead - cell interactions. The experiments were performed in the presence of 5 mM Mg²⁺ plus EGTA at room temperature. For 2 s contacts each bar represents data from 10 cell-bead pairs from each of three donors (total of 30 cell-bead pairs). Each cell - bead pair was contacted 25 times. For 60 s contacts, n = 105 from seven donors. The surface concentration of VCAM was ∼650 sites per μm². Error bars indicate standard error. The stars denote a statistically significant difference in adhesion probability (^∗∗p < 0.01 and ^∗∗∗p < 0.001, Student's t-test).

Dependence of adhesion probability on contact time. The experiments were performed at room temperature as repeated tests of cell-bead touches for different contact durations. The total adhesion probability was converted to the expected number of bonds 〈n〉 (see Eq. 1) and plotted as 〈n〉 versus time. Error bars indicate standard error. (A) The experiments were performed in the presence of 5 mM Mg²⁺ plus EGTA for 1, 2, 5, 10, 20 and 60 seconds duration. The model used to fit the upper data was Eq 2 plus a linear portion reflecting background adhesion: 0.0055x + 0.089. From nonlinear least squares regression, 〈n〉_o = 0.19 ± 0.035, τ_RZ = 14.8 ± 4.09 s, and K_DRZ = 53.7 ± 3.67. (B) The experiments were performed in 3 mM Mn for 1, .2, .5, 10, and 20 seconds duration. Model used to fit the data was Eq 2 with a linear portion reflecting background: 0.137 + 0.021x. The fitted parameters were 〈n〉_o = 0.29 ± 0.418, τ_RZ = 3.21 ± 2.894 s, and K_DRZ = 23.25 ± 4.533. (C) The experiments were performed in the presence of 1.5 mM Ca²⁺ for 1, 2, 5, 10 and 20 seconds duration. Model used to fit the data was Eq 2 with a linear portion reflecting background: 0.039 + 0.0067x. The fitted parameters were 〈n〉_o = 0.0 (fixed), τ_RZ = 2.33 ± 1.15 s, and K_DRZ = 160 ± 23.

Effect of blocking antibodies on adhesion to VCAM-coated beads. (A) Every bar represents the adhesion probability to VCAM-1 coated beads for 10 second contacts. The amount of VCAM is 650 sites/μm². Addition of anti-β₁ blocking antibody 4B4 caused the 70% reduction in adhesion probability in all buffers tested in the study: 1.5 mM Ca²⁺, 5 mM Mg²⁺, 3 mM Mn²⁺. (B) Each bar represents 30 cell - VCAM-bead pairs interacting for 2 seconds (25 contacts for each pair) in the presence of 5.0 mM Mg²⁺ plus EGTA at room temperature. The amount of VCAM is 650 sites/μm². The β₂ blocking antibody IB4 had no effect on the adhesion probability, but the β₁ blocking antibody 4B4 reduced the adhesion probability to 15%. The slight additional degrease in P_adh when 4B4 and IB4 were used in combination was not statistically significant. (C and D) Experiments were performed as repeated tests of 20 contacts for each cell-bead pair. Contact time was 20 seconds. Each bar represents data from 8 to 36 cells total. Gray bars represent control condition for the sparsed bars (condition labeled on the x axis). (C) In the presence of 5 mM Mg²⁺ plus EGTA; (D) In the presence of 1.5 mM Ca²⁺. Error bars represent standard error. The asterisk denotes a statistically significant difference in adhesion probability (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001; Student's t-test).