Leukotriene B4 activates intracellular calcium flux. (a) Intracellular calcium flux measurements in human peripheral blood mononuclear cells (PBMCs) cultured with macrophage colony-stimulating factor (M-CSF) for 8 days, labeled with fluo-4 AM, pretreated with either dimethyl sulfoxide control, 100 nM BLT1 and BLT2 antagonist cocktail, 1 μM phospholipase C (PLC) inhibitor U73122 or 100 μM calcium release–activated channel (CRAC) inhibitor 2-aminoethoxydiphenyl borate for 15 minutes prior to imaging in real time followed by acute activation with 10 nM LTB4. Representative data from three experiments are shown. The scale bars represent 10 μm. (b) Normalized fluo-4 AM intensity plotted for representative cells over the 600-second time course. (c) Graphical representation of the percentage of cells fluxing calcium in human PBMCs cultured with M-CSF for 8 days, labeled with fluo-4 AM and acutely activated with either 100 ng/ml receptor activator of nuclear factor κB ligand (RANKL), 10 nM LTB4 or both simultaneously. Representative data and images from three experiments are shown. *P < 0.05.