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To measure calcium mobilization upon receptor triggering, we preloaded mast cells (2 × 106 cells per mL) with 0.5 μg/mL IgE–anti-DNP antibody for 4 h in medium without IL-3 and then loaded with 3 μM Fluo-4 AM (Molecular Probes) calcium indicator for 30 min at 37 °C. Calcium flux was induced by adding HSA-DNP (30 ng/mL) and ionomycin (2 μM) and measured by flow cytometry. Analysis of the kinetics of changes in intracellular calcium concentration in different experiments was performed exactly as described (61).
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