Enlarged lysosome-related compartments but normal phagocytic uptake in Lyst-mutant cells. (A, left) WT and Bg-J BMDCs were stained for the indicated endosomal/lysosomal marker proteins. Localization of endogenous proteins was analyzed by confocal microscopy. Bars, 5 µm. (Right) The mean size of the indicated particles was quantified by using ImageJ software (1.50c). In the graphs, each dot represents mean particle size in one cell. Lines show total mean ± SEM. *, P < 0.05 (Mann-Whitney U test). (B) BMDCs from WT and Bg-J mice were preincubated with LysoTracker red and Lysosensor green. Fluorescence images were generated from live cells by confocal microscopy. Bars, 10 µm. DIC, differential interference contrast. (C) Phagocytic uptake of fluorescence-labeled E. coli by WT and Bg-J BMDCs was assessed by flow cytometric analysis at the indicated times. WT, shaded; Bg-J, red lines. (D) WT and Bg-J BMDMs were exposed to E. coli particles labeled with the pH-sensitive dye pHrodo. Phagosomal acidification was assessed by fluorescence microscopy after 40 min of incubation. pH sensitivity was controlled by analysis of pHrodo-labeled E. coli particles in buffers with different pH (bottom). Bars, 10 µm. (E and F) WT and Bg-J BMDMs were exposed to E. coli particles double labeled with the pH-sensitive dye pHrodo and with eFluor 670. Particle uptake and phagosomal acidification were assessed by flow cytometric analysis. (E) Flow cytometric profiles at 0 min (top) and 90 min (bottom) of incubation showing fluorescence staining for pHrodo versus eFluor 670. (F) Flow cytometric quantification of pHrodo-positive cells at the indicated time points. As specificity control, cells were incubated with 30 mM NH₄Cl to quench lysosomal acidification, or cells were kept on ice. Data are mean ± SD from two independent cultures. All data are representative of at least two independent experiments.