Prolonged Ag survival and slow endosomal acidification in IFN-DCs after OVA uptake. (A-B) Time course analysis by flow cytometry of Ag uptake and processing in DCs. Cells were pulsed with OVA-FITC (A) or DQ-OVA (B) for 15 minutes at 37°C and then chased with medium for the indicated intervals. A representative experiment of 5 is shown. The mean fluorescence values are indicated in each panel. (C) Detection by CLSM examinations (central optical sections) of cytoplasmic acidic vesicles in unfixed DCs stained with LysoSensor Yellow/Blue DND-160 (pseudocolor gray). Scale bars represent 10 μm. (D) Flow cytometry profiles of unfixed DCs stained with LysoSensor Green DND-189. (E) Time course analysis by flow cytometry of LysoSensor Green DND-189 fluorescence changes (mean ± SD of 3 independent experiments) in DCs allowed to take up unconjugated OVA for the indicated times. (F) NOX-2 and Rac2 detection by CLSM examinations of DCs, fixed, permeabilized, and stained with anti–NOX-2 or anti-Rac2 Abs (pseudocolor gray). Nuclei are reported in blue (4′, 6-diamidino-2-phenylindole). Scale bars represent 10 μm. Panels are representative of 5 (NOX-2) or 3 (Rac2) independent experiments. (G) Western blot analysis (1 of 3) on total cell lysates from DCs that were allowed to internalize OVA for 15 minutes and then were washed and chased for 2 or 18 hours. Blots were incubated with anti–NOX-2 and anti-OVA Abs. Actin was used as quantitative loading control.