Phagolysosomes do not undergo exocytosis but fragment. (A) 1 h after phagocytosis of IgG-opsonized beads, RAW cells were treated with 10 µM ionomycin in the presence of 1.2 mM of Ca²⁺, and live-cell DIC images were acquired at the indicated time points. Scale bars: 10 µm. (B) Number of latex beads per cell at the indicated treatment times, normalized to the number of beads/cell observed at the onset of the treatment (T₀). Data are mean ± SEM of 3 independent experiments, where 10 cells were measured in each experiment. (C) RAW cells expressing PLCδ1-PH-GFP and containing IgG-opsonized beads in phagosomes were treated with 10 µM ionomycin or DMSO (control) in the presence of 1.2 mM of Ca²⁺. Images were acquired before or after 10 min of treatment. Asterisks correspond to the positions of the beads, as shown in the insets. Scale bars: 10 µm. (D) Live-cell imaging time series of IgG-aggregated fluorescent 0.1-µm latex beads after 2-h phagocytosis by RAW cells. Beads are depicted in a rainbow scale. Red and blue correspond to the highest and lowest florescence intensity levels, respectively. The smaller panels show the far red and DIC channels for the first frame. The cell contour is delineated with white dashes. See corresponding Video 4. Scale bars: 5 µm. (E) Macrophages were assessed for exocytosis of digested phagosomal contents. RAW macrophages internalized mRFP1-labeled E. coli for 1 h and then were incubated for the indicated time points. TCA precipitates of cell media and cell lysates were probed for mRFP1, detecting a native and a cleaved mRFP1 product that accumulated over time. GAPDH was used as a loading control and to detect cell lysis. (F) Quantification of cleaved mRFP1 levels normalized to GAPDH in media and within cells. Data are shown as mean ± SD of six independent experiments. Conditions were compared using one-way ANOVA and Holm-Sidak’s post hoc test (*, P < 0.05).