Functional effects of Ca_o²⁺ on B cells. (A,B) Early activation: PBMCs were cultured in PBS plus or minus CaCl₂ (500 μM) for 4 hours, prior to being washed and recultured in AIM-V with or without IL-2 (1000 U/mL) plus S28690 (1 μg/mL) (2/S) or anti-Ig (10 μg/mL). After 2 hours of stimulation, cells were incubated with DCFH-DA and CD19 antibodies for 20 minutes prior to flow cytometric analysis (A). *P < .05; n = 3. After 4 hours of stimulation, cells were stained with antibodies against CD86, 41BBL, and CD19 (B). (C,D) Cytokine production: Isolated B cells were cultured in PBS plus or minus 500 μM CaCl₂ prior to being washed and recultured in AIM-V with or without IL-2 plus S28690 for 24 hours. P-STAT1, P-STAT3, and β-actin (loading control) levels were then determined by immunoblotting (similar results were obtained using samples from 3 different donors) (C). TNF-α was measured by cytometric bead array (D). *P < .005; n = 4.