Role of intracellular and extracellular calcium in fMLP- and SDF-1–induced LDV-FITC binding. U937-FPR cells pretreated with either DMSO (0.1%, 30 minutes) or BAPTA-AM (12 μM, 30 minutes) were stimulated at time 0 with Mn²⁺, buffer, thapsigargin (Tg; 1 μM), fMLP, or SDF-1. Intracellular calcium levels (Fluo-3 fluorescence; A) and LDV-FITC binding (B) were analyzed in real time by flow cytometry. One of 4 independent experiments is shown. (C) Illustrates LDV-FITC binding induced by ionomycin (0.1 and 1 μM) compared with Mn²⁺, fMLP, and buffer control. (D) Single-cell calcium imaging of Fura-2–loaded U937-FPR cells stimulated with fMLP in the presence of DMSO (control), SKF96365 (100 μM), or EGTA (5 mM). (E) LDV-FITC binding to U937-FPR cells pretreated with SKF96365 prior to stimulation (arrow) with fMLP, SDF-1, Mn²⁺, or thapsigargin. One of 4 independent experiments is shown. (F) Basal LDV-FITC binding (mean ± SEM) to U937-FPR cells in assay buffer containing the indicated concentrations of Mg²⁺ and Ca²⁺ (left). U937-FPR cells were stimulated (arrow) with fMLP, SDF-1, or Mn²⁺ and analyzed for LDV-FITC binding in a calcium-free assay buffer containing 1 mM Mg²⁺ (right). One of 3 independent experiments is shown.