Soluble E-selectin prolongs PAF-induced Ca²⁺ mobilization. (A) Freshly isolated neutrophils were loaded with Fura2-am (2 μM) for 30 minutes at 37°C in Ca²⁺- and Mg²⁺-free HBSS, then washed and resuspended in HBSS containing Ca²⁺ and Mg²⁺, and then preincubated with or without soluble E-selectin (E-sel, 5 μg/mL) for 15 minutes at 37°C as indicated. For blockade of CD62E, ENA2 (1:50) and soluble E-selectin were preincubated for 1 hour before being added to neutrophils. Neutrophils were stimulated with PAF (100 nM) after recording “baseline” Ca²⁺ levels for 60 seconds. Data are shown as a representative trace of 5 separate experiments showing similar results and are expressed as percent of peak [Ca²⁺]_i following PAF stimulation (typically from 50 nM control levels to 2.5 μM following stimulation). (B) Traces from panel A have been integrated to calculate area under each curve using GraphPad Prism software (GraphPad, San Diego, CA) to compare the effects of various inhibitors. Data from 5 separate experiments that were performed and expressed as mean ± SEM. *Statistically different (P < .05) from PAF-treated controls.