Plasmacytoid DCs are not responsive to whole bacteria. (A) pDCs and mDCs cultured in isolation (■) or together (▨) in presence of fixed S aureus bacterial particles, and the percentage of cells expressing CD40 was determined. A representative experiment of 3 is shown, and similar results were obtained using other bacteria (data not shown). (B) TNF-α production was measured in the culture supernatant of pDCs (□) and mDCs (■) cultured in isolation or together (▩) in the presence or absence of fixed S aureus. A representative experiment of 3 is shown. (C) Phagocytosis of fluorescently labeled E coli fixed bacterial particles by CD16 and CD1c mDC subsets or pDCs was measured by flow cytometry in the presence (left panels) or absence (right panels) of cytochalasin D (Cyt D). Similar results were obtained performing the experiments at the time points of incubation, as follows: 15 and 30 minutes; 1, 4, 6, and 8 hours; and overnight (data not shown). One representative experiment of 5 is shown. (D) Uptake of fluorescently labeled S aureus bacterial particles by pDCs pretreated for 4 hours with the indicated stimuli. Similar results were obtained after overnight pretreatment of pDCs. A representative experiment of 5 is shown. (E) Fold induction of TNF-α production measured in the supernatant of mDCs (■) or pDCs (□) cultured in presence of live growing E coli, Y pestis (pLCR-mutant strain), and GBS. One representative experiment of 3 is shown.