Figure 5.

Tethering and rolling on P-selectin are prerequisites for SDF-1–triggered firm arrest of CD34⁺ cells on ICAM-1–containing surfaces. (a) CD34⁺ cell and T lymphocyte tethering to various adhesive substrates containing P-selectin, SDF-1, and ICAM-1 at a shear stress of 1 dyn/cm². Motions of individual cells tethered to the different substrates were monitored over a 45-second period, and were divided into 3 categories as described in Methods. The fraction of each category within each experimental group (i.e., rolling, rolling-associated arrests, and immediate arrests) is presented in the stacked bars. The categories were analyzed only for cells that remained bound to the substrate at a shear stress of 2.5 dyn/cm². (b) Resistance to detachment by incremented shear stresses of CD34⁺ cells accumulated at 1 dyn/cm² on P-selectin/ICAM-1. The absolute numbers of cells accumulated during 1 minute at 1 dyn/cm² and the number of cells remaining bound at the end of a 5-second interval of each shear increment are depicted. The percentage of stationary (arrested) cells within the cells remaining bound at a median shear stress (7.5 dyn/cm²) is shown in parentheses near the data points for each experimental group. ICAM-1 and SDF-1 were coated at 0.4 μg/mL and 10 μg/mL, respectively. P-selectin/ICAM-1 spots were prepared by mixing P-selectin and ICAM-1 in PBS/1% octyl glucoside and diluting the mixture in coating medium to final concentrations of 1 μg/mL and 0.5 μg/mL, respectively. Substrates were washed and then coated with SDF-1 as described in Methods. To assess the effect of soluble SDF-1, cells were preincubated in binding medium containing 1 μg/mL SDF-1 for 1 minute and perfused unwashed over the P-selectin/ICAM-1 substrate. Results shown in a and b are presented as mean of 2 determinations ± range. Sol., soluble; Imm., immobilized.