Effects of Ca_o²⁺ on Ca_i²⁺ release in human B cells. AIM-V, RPMI with or without calcium, or PBS (500 μL) was added to Indo-1 AM–labeled PBMCs bathed in 750 μL Ca²⁺-free PBS (A), or 500 μM CaCl₂ was added to cells bathed in PBS containing various levels of calcium (B-D). Alternatively, MgCl₂ (C) or a TLR-7 agonist, S28690 (1 μg/mL), (D) was added to other Indo-1–loaded cells in Ca²⁺-free PBS. indicates addition of the stimulus during the 6-minute collection period of calcium flux data. Using Indo-1 AM, bound Ca²⁺ is detected at approximately 400 nM (blue) and free Ca²⁺ is detected at approximately 475 nm (violet). The data are presented as the ratio of Indo violet/Indo blue as a function of time for the B-cell population (gated on CD19⁺ cells) and represent 8 independent experiments.