Effects of CD157 cross-linking on intracellular Ca²⁺ concentration. Purified PMNs were first incubated with murine IgG Fc fragments (150 μg/mL) at 4°C for 15 minutes to block binding of the primary mAb to Fc receptors,¹⁷  then loaded with fluo3-AM; after calibration of the baseline fluorescence, the different stimuli were added (arrow) and cell events acquired. Dynamic changes in [Ca²⁺]_i were monitored continuously by plotting the shift in the fluo3-AM fluorescence over a 516-second time course. Data are representative of 3 separate experiments. (A) Top panel: PMNs were treated with saturating amounts of anti-CD157 Mo5 mAb (5 μg/mL) and increasing concentrations (20 μg/mL-100 μg/mL) of F(ab′)₂-RaMIgG. Bottom panel: a significant increase in [Ca²⁺]_i was observed following incubation with fMLP (100 nM), whereas a control isotype-matched anti-CD3 mAb did not elicit any Ca²⁺ currents. (B) The mobilization of Ca²⁺ after CD157 cross-linking (top) or fMLP stimulation (bottom) was monitored after treatment for 5 minutes with 3 mM EGTA. (C) Dye-loaded PMNs were preincubated in the presence or absence of 100 μM 8-Br-cADPR and then stimulated with Mo5 mAb and F(ab′)₂-RaMIgG or 100 nM fMLP (arrow). (D) The effects of CD157 and CD18 cross-linking or fMLP stimulation were analyzed in PMNs pretreated with 100 nM wortmannin or 500 nM staurosporin. Data are representative of 3 experiments with cells from different donors.