the correct focal plane, tethers could be directly and definitively visual- ized. Results Tethering of Neutrophils to Spread Platelets Neutrophils were perfused over surfaces sparsely coated with platelets at wall shear rates of 50–300 s21 to deter- mine whether, under physiological flow conditions, the ini- tial adhesive interactions between free-flowing neutro- phils and immobilized platelets can lead to the formation of elongated membrane tethers. During perfusion, elon- gated membrane tethers were pulled from neutrophils af- ter capture by spread platelets (Fig 1 a). The process of tether formation caused a slight teardrop-shaped deforma- tion of the neutrophil (Fig. 1, b and c). In real-time, the tethering process was easily distinguished from a rolling or pausing neutrophil by identification of surface features on the neutrophil that did not rotate as the neutrophil trans- lated downstream from the platelet at a velocity consider- ably slower than the flow velocity (Fig. 1 b). Fig. 1 b shows a free-flowing neutrophil traveling at 1,285 mm/s (wall shear rate 5 200 s21) being captured by an adherent plate- let, forming a 2.8-mm-long tether, and then releasing after 113 ms. Fig. 1 c shows an example of an extremely thin tether formed between a neutrophil and a platelet. For the neutrophils that formed tethers, determination of the cell center position with time demonstrated the rapid approach velocity of a neutrophil in a streamline above the platelet, the extremely rapid deceleration dur- ing capture by the platelet, tether extension, and the acute release of the neutrophil into a streamline closer to the surface (Fig. 2). In Fig. 2 a, the cellular motion of the colli- sion event imaged in Fig. 1 b demonstrated that the upstream velocity of neutrophil was uniform before the adhesive event, whereas the downstream velocity after de- tachment was markedly slower but almost instantaneously obtained after release. Fig. 2 b shows the distance traveled by the neutrophil as it approached the spread platelet, as a membrane tether grew, and finally as the tether detached from the stationary platelet, and the neutrophil translated Figure 2. Motion of a free- flowing neutrophil that teth- ers to a stationary platelet. (A) The x-y position of the cell centroid of the neutro- phil shown in Fig. 1 b, before, during, and after the neutro- phil has formed a membrane tether with the adherent platelet. (b) The distance traveled with time by the neutrophil relative to its en- trance point. The slopes of the solid lines represent the average velocity for each stage: the approach velocity, tether growth velocity, and the release velocity. The Journal of Cell Biology, Volume 149, 2000 722 downstream (with observable rotation of surface fea- tures). The slopes of the lines drawn in Fig. 2 b represent the average velocity of each of these stages, and they dem- onstrate the distinct transitions between each phase of ap- proach, tether growth, and release. Effect of Wall Shear Rate on Neutrophil–Platelet Membrane Tether Growth To gain insight into the strength and the rate of growth of the tethers formed between neutrophils and platelets, the influence of wall shear stress on tether growth rate, length, and duration was studied. Wall shear rates ranging from 50 to 300 s21 (tw 5 0.5–3.0 dyne/cm2) were investigated by varying the flow rate of the perfusion solution through the chamber. The average tether growth rate increased signifi- cantly (P , 0.001) from 6 to 40 mm/s as the wall shear rate was increased from 100 to 250 s21 (Fig. 3 a), whereas the average time of tether duration decreased significantly (P , 0.001) from 630 to 133 ms with an increasing wall shear rate (Fig. 3 b). Although the average tether length appeared to go through a maximum at 200 s21 (Fig. 3 c), these values were not statistically different with the overall average tether length being 5.9 6 4.1 mm (range 1–25 mm, n 5 63 tethers). At wall shear rates below 50 s21 and above 300 s21, transient tethers were not detected. For neutrophil collisions with spread platelets involving tether formation, there is a movement of neutrophil posi- tions with gap separations that are about a micron away from the surface to positions with gap separations ,0.3 mm (Fig. 4). Fig. 4 a is a plot of the average free-flowing neutrophil velocity before and after tethering, with the sta- tionary platelet as a function of wall shear rate. As ex- pected, both the approach velocity and release velocity in- creased with an increasing wall shear rate. By using the measured approach and release velocities, we calculated the distance between the neutrophil (effective hydrody- namic radius, R 5 4.25 mm) and the wall using the theoret- Figure 3. Effect of wall shear rate on (a) tether growth rate, (b) tether duration, and (c) tether length. Data points are the average 6 SD of n measurements at each shear rate (gw 5 100 s21, n 5 9; gw 5 150 s21, n 5 21; gw 5 200 s21, n 5 23; and gw 5 250 s21, n 5 10).