Effect of soluble E-selectin is time-dependent. (A) Freshly isolated neutrophils were loaded with Fura2-am (2 μM) as for Figure 1 and preincubated with E-selectin (5 μg/mL) for various times as indicated. The cells were then stimulated with 100 nM PAF after 60 seconds of recording. Data from 3 separate experiments that were performed are expressed as mean area under the curve ± SEM. *Statistically different (P < .05) from PAF-treated controls. (B) Freshly isolated neutrophils were preincubated with soluble E-selectin and samples were either washed to remove E-selectin (WO) or used after 15 minutes of incubation. Data shown are expressed as mean area under the curve ± SEM from 3 independent experiments.