Three distinct monoclonal antibodies were tested for their capacity to deplete pDCs. Two of the antibodies are specific for B220: Rat anti-B220 IgG2a and Rat anti-B220 IgM; and one is specific for Siglec-H: Rat anti-Siglec-H IgG2b. The latter antibody is an isotype shared by other established depleting antibodies such as anti-CD4 (clone GK1.1) and anti-Gr1 (clone RB685C). (A) At high concentrations of 1 mg, none of the antibodies alone, or in combinations of two, depleted pDCs at 24 hr, instead the antibodies masked the targeted antigen as illustrated in flow plot overlays of pDCs identified by Ly6C and Ly6D from all splenic CD19 cells. Bottom row, flow cytometry overlays of all CD19 cells (yellow) and gated pDCs (blue) illustrate the masking of B220 and SiglecH 24 hr after muMT mice were ip injected, alone or in combination, with anti-mouse B220 rat IgG2a, anti-mouse B220 rat IgM, and anti-mouse Siglec-H. (B) However, when all three antibodies were given, pDCs were diminished by 63–83% in the spleen and LNs of muMT mice, and this depletion persisted across 24, 48, and 72 hr. Top row, gated Ly6C+Ly6D+ pDCs from all splenic CD19 cells. Bottom row, flow cytometry overlays of all CD19 cells (yellow) and gated pDCs (blue) illustrating the few cells remaining at 24, 48, and 72 hr after pDC-depleting triple antibody (Ab) treatment (anti-mouse B220 rat IgG2a, anti-mouse B220 rat IgM, and anti-mouse Siglec-H). Scatter plot represents four independent experiments, mean ± SEM. pDC, plasmacytoid dendritic cell.