The activation threshold is higher for monocytes than for pDCs. Fresh human PBMCs were incubated for 4 hours with 10 μg/mL fluorescent spheres (A) or 5, 10, 20, or 30 μg/mL fluorescent spheres (B) stained by APC-labeled CD11c and analyzed by fluorescence-activated cell sorting. The dot plots presented in panel A and analyzed in panel B were gated on monocytes on the basis of forward-side light scatter plot (gate r9, supplemental Figure 6A, or gate r3, supplemental Figure 6B). The value in each quadrant in panel A indicates the percentage of cells as a percentage of total cells. In panel B, mean fluorescence (FL1 for 200 nm, FL2 for 500 nm, and FL3 for 1000 nm) of CD11c⁺ cells at different particle concentrations is shown. (C) TNF-α and interferon-α present in the supernatant of fresh human PBMCs incubated 24 hours with 15, 20, 25, 30, or 35 μg/mL protamine RNA nanoparticles (220 nm) was tested by ELISA. The data shown are representative of 3 independent experiments.