Effects of Ca_o²⁺ on CD83 expression on human B cells. (A) Flow cytometric analysis of CD83 in monocytes, T cells, and B cells (indicated by costaining with CD14, CD3, and CD19 antibodies, respectively) immediately after isolation (0 h) and 4 hours after culture in AIM-V. The example shown is representative of 8 different experiments. (B) PBMCs were cultured in different media (PBS without Ca²⁺ or Mg²⁺, HBSS with Ca²⁺ and Mg²⁺, AIM-V, DMEM, αMEM, DMEM + F12, or RPMI) for 4 hours and then analyzed by flow cytometry. Statistical analysis was performed on percentage CD83 expression of samples cultured in media compared with baseline. *P < .001. (C) PBMCs were cultured in RPMI with and without calcium, with AIM-V alone, or with AIM-V and EDTA (10 μM) or BAPTA (10 μM) for 4 hours prior to flow cytometric analysis of the B cells (gated on CD19⁺ cells). The results were similar for 6 additional samples. (D) CD83 on gated B cells from PBMCs cultured in calcium-free PBS (left panel) or AIM-V (right panel) and treated with EDTA, S28690, or EDTA + S28690 for 4 hours prior to analysis by flow cytometry. (E) PBMCs were cultured in PBS alone, with MgCl₂ (50, 250, 500, or 1000 μM) or with CaCl₂ (5, 50, 250, 500, 1000 μM) for 4 hours, and then B cells were analyzed by flow cytometry. The averages and standard errors of the percentage of cells expressing CD83, and the mean fluorescent intensity (MFI) of expression, from the indicated numbers of samples, are shown. The statistical significance of the differences between percentage CD83 expression of MgCl₂- or CaCl₂-treated compared with untreated samples is indicated. *P < .05; **P < .001; n = 5.