Figure 7.
Phagosome (Phago.) maturation consumes free lysosomes. (A and C) Macrophages were challenged with IgG beads (A) or mCherry-Lp (C) and stained for LAMP1 and Lp. Images show LAMP1 (A and C) and Lp (C). Scale bars: 10 µm (A), 5 µm (C). (B) Number of free lysosomes per cell at specified times after phagocytosis (Time AP) for images described in A. (D) Number of free lysosomes per cell at specified times after phagocytosis (Time AP) for images described in C. In B and D, data are mean ± SEM of 3 independent experiments, where 60 cells (B) and 20 cells (D) were quantified per condition in each experiment, normalized to resting cells, and tested by one-way ANOVA with Tukey’s test (*, P < 0.05). (E) RAW cells that engulfed Lp were chased 6 h to allow for Lp fission and presented for 1 h with IgG-opsonized beads (second round of phagocytosis). Cells were fixed and stained for Lp. The arrow shows bacterial debris in the bead phagosome. Scale bars: 5 µm (main panels), 0.5 µm (insets). (F) Percentage of beads that were positive for Lp fragments in E. Data are mean ± SEM of 3 independent experiments with 20 cells quantified for each condition per experiment. (G) Macrophages were presented with mRFP1–E. coli for 7 h as a first round of phagocytosis before being challenged with ZsGreen–E. coli for a second round of uptake. As a control, resting cells were challenged only with one round of phagocytosis using ZsGreen–E. coli. Scale bars: 10 µm (main panels), 2 µm (insets). (H) Quantification of mean mRFP1 fluorescence intensity (derived from first-round phagosomes) on ZsGreen–E. coli-containing phagosomes (second-round phagosomes) as described in G. Data were normalized to macrophages without the first wave of phagocytosis and presented as mean ± SD of 148–348 cells across 3 independent experiments. Conditions were compared statistically using one-way ANOVA with Tukey’s test (**, P < 0.01; ***, P < 0.001).  Dashed lines show cell contours (A, C, E, and G).

Phagosome (Phago.) maturation consumes free lysosomes. (A and C) Macrophages were challenged with IgG beads (A) or mCherry-Lp (C) and stained for LAMP1 and Lp. Images show LAMP1 (A and C) and Lp (C). Scale bars: 10 µm (A), 5 µm (C). (B) Number of free lysosomes per cell at specified times after phagocytosis (Time AP) for images described in A. (D) Number of free lysosomes per cell at specified times after phagocytosis (Time AP) for images described in C. In B and D, data are mean ± SEM of 3 independent experiments, where 60 cells (B) and 20 cells (D) were quantified per condition in each experiment, normalized to resting cells, and tested by one-way ANOVA with Tukey’s test (*, P < 0.05). (E) RAW cells that engulfed Lp were chased 6 h to allow for Lp fission and presented for 1 h with IgG-opsonized beads (second round of phagocytosis). Cells were fixed and stained for Lp. The arrow shows bacterial debris in the bead phagosome. Scale bars: 5 µm (main panels), 0.5 µm (insets). (F) Percentage of beads that were positive for Lp fragments in E. Data are mean ± SEM of 3 independent experiments with 20 cells quantified for each condition per experiment. (G) Macrophages were presented with mRFP1–E. coli for 7 h as a first round of phagocytosis before being challenged with ZsGreen–E. coli for a second round of uptake. As a control, resting cells were challenged only with one round of phagocytosis using ZsGreen–E. coli. Scale bars: 10 µm (main panels), 2 µm (insets). (H) Quantification of mean mRFP1 fluorescence intensity (derived from first-round phagosomes) on ZsGreen–E. coli-containing phagosomes (second-round phagosomes) as described in G. Data were normalized to macrophages without the first wave of phagocytosis and presented as mean ± SD of 148–348 cells across 3 independent experiments. Conditions were compared statistically using one-way ANOVA with Tukey’s test (**, P < 0.01; ***, P < 0.001).  Dashed lines show cell contours (A, C, E, and G).

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