FIG. 1.

Analysis of antigen-processing in Salmonella-infected BM-DC. BM-DC from C56BL/6 mice were infected at an MOI of 25 with Salmonella WT or a strain deficient in sseC, encoding a translocon subunit of the SPI2-T3SS (SPI2) for 1 h, were stimulated by addition of 1 μg of LPS/ml or remained uninfected (mock). In addition, BM-DC were stimulated with fluorescently labeled endocytotic marker Texas Red-OVA (A and B) or DQ-OVA (C and D). Texas Red-OVA fluorescence (red) decreases with increased proteolytic degradation, while fluorescence of DQ-OVA (green) is induced by proteolysis. At the indicated time points after infection, cells were fixed and subjected to immunofluorescence analyses after staining for CD11c (blue) and Salmonella (green in panel A, red in panel C). For quantification of antigen degradation, Salmonella-infected, LPS-stimulated or uninfected BM-DC were subjected to flow cytometry and gated on the CD11c-positive population. The MFI and standard deviation for Texas Red fluorescence were determined at various time points of infection (B), and the MFI for DQ-OVA was determined at 16 h after infection (D). The data are representative of three analyses with similar outcomes. *, P < 0.05; ns (not significant), P > 0.05. Scale bar, 10 μm.