CD300a down-regulates BCR-mediated activation signals. (A) Purified human B cells (upper panel) or DT40 cells expressing CD300a (lower panel) were loaded with Fluo-4 and Fura-Red. Then, cells were acquired in a flow cytometer and stimulated with anti-BCR plus anti-CD300a mAb (gray line) or anti-BCR plus isotype control (black line), according to the protocol described in “Calcium mobilization assays.” Intracellular Ca²⁺ concentration was measured by the ratio of Fluo-4/Fura-Red as a function of time. Results are representative of 2 (human B cells) and 4 (DT40 cells) independent experiments. (B) DT40 cells expressing CD300a were transiently transfected with a NFAT luciferase reporter plasmid and stimulated with medium, antichicken BCR plus isotype control, or antichicken BCR plus anti-CD300a mAb as indicated. The measured luciferase activity was normalized to the activity obtained for the treatment with phorbol myristate acetate plus ionomycin. The data presented are the mean plus or minus SEM for 6 separate experiments. (C) Purified human B cells were transfected with nontarget or CD300a siRNA. Then, cells were stimulated with anti-Ig (IgG/A/M, IgM, or IgG) and CpG, and the proliferation was measured after 4 days of culture as described in “Cell cultures.” A representative donor is shown. Numbers in the upper left quadrant indicate the percentage of CD22⁺ cells that have divided one or more times (upper panel). Graphic representation of proliferating cells treated with CD300a siRNA normalized to the nontarget siRNA-treated cells (lower panel, left side) and the CD300a mRNA relative levels measured 4 days after transfection with siRNA (n = 3) are shown (lower panel, right side).