Ro5-4864 suppresses Ag-triggered Ca2+ flux. (A) BMMCs were pretreated for the indicated times with 100 μM Ro5-4864 or for 30 min with a corresponding amount of DMSO and then either stimulated with Ag (DNP-HSA) (open symbols) or left unstimulated (solid symbols) for another 10 min. Degranulation was measured by β-hexosaminidase assay. Each value is the mean of duplicates ± SEM. (B) Intracellular Ca2+ was measured in BMMCs by flow cytometry using the Ca2+-sensitive fluorescent dyes fluo-3 and fura red. Steady-state fluorescence was determined for 1 min before 1 mM EDTA (first arrow) was added for 1 min to chelate extracellular Ca2+. Denoted BZDs or DMSO were added (second arrow) and incubated for 2 min. Cells were then stimulated with 200 ng/ml Ag (third arrow) and the resulting Ca2+ response derived from intracellular store depletion was measured for 3 min. Finally, 2 mM CaCl2 was added (fourth arrow) to replenish extracellular Ca2+ stores and the resulting SOC influx was measured for 2 min. Comparable results were obtained with cells from different cultures (n≥3). (C) Measurements were performed as described under (B) with the exception that cells were not stimulated with Ag, but with the SERCA inhibitor thapsigargin (TG). Comparable results were obtained with cells from two different cultures.