Internalization of B Cell Receptors in Human EU12 μHC+ Immature B Cells Specifically Alters Downstream Signaling Events
Anti-IgM antibody induces BCR internalization in EU12 μHC+ immature B cells and in mature Daudi or Ramos B cells. (a) Human U12 μHC+, Daudi, and Ramos cells were treated with goat anti-IgM antibody F(ab′)2 fragments (2 μg/mL) for different times. The cells were stained with the Cy5 labeled anti-IgM antibodies at different time points and subjected to flow cytometric analysis. The percentages of cells expressing cell surface IgM are indicated. (b) Murine 70Z/3 cells were treated with goat anti-IgM antibodies (5 μg/mL) for different times; cell surface IgM were monitored by APC labeled anti-IgM antibodies. The percentages of cells expressing cell surface IgM are indicated. Unstained samples were used as negative control. (c) Immunofluorescence analysis. For untreated cells, EU12 μHC+, Daudi, or Ramos cells were fixed and stained with Cy5 labeled anti-IgM antibodies. For anti-IgM antibody treated cells, EU12 μHC+, Daudi, and Ramos cells were treated with Cy5 labeled anti-IgM (2 μg/mL) for 30 min. Cells were then fixed and visualized on glass slides with fluorescence microscopy. (d) Human EU12 μHC+ or Daudi cells were treated with anti-IgM antibodies (2 μg/mL) for 24 hours and cell surface HLA and CD86 levels were analyzed by flow cytometry.