Figure 6.
Phagolysosome fusion and intracellular killing by thrombocytes and other leukocytes are shown. (A) Goldfish phagocytic thrombocytes stained with fluorescent dextran and incubated with non-fluorescent latex beads measuring 3 μm in diameter for 3 h at 25°C. Thrombocyte alone (upper), with bound (middle), or ingested (lower) beads. (B) GB10 mAb- goldfish phagocytes with bound beads (upper) and ingested beads (lower) and a phagolysosome ring (arrow). A phagolysosome ring is observed around the ingested particle (arrow). Dextran, FITC–dextran; GB10, mAb for goldfish thrombocytes; DRAQ5, nuclei. (C,D) Common carp phagocytic thrombocytes stained with LysoTracker Red to visualize acidic organelles. (C) Thrombocytes with bound (upper) or ingested (lower) non-fluorescent latex beads measuring 3 μm in diameter. (D) Phagocytic mAb- leukocyte with an ingested bead (arrow). The phagolysosome ring as detected by acidic probe staining. LT (red), acidic organelles visualized by LysoTracker Red; HB8 (green), HB8 mAb specific for common carp thrombocytes; DAPI (blue), nuclei stained with DAPI. Original magnification, ×400. Data are representative of three independent experiments. (E,F) Intracellular bactericidal activity of thrombocytes. (E) Flow cytometry of phagocytic common carp thrombocytes incubated with GFP-expressing live E. coli for 3 h at 25°C. The number indicates the percentage of phagocytic cells in HB8+ thrombocytes. Data are representative of three independent experiments. (F) Survival rate of internalized bacteria counted on Luria–Bertani agar plates. Data are expressed as the average of three independent experiments, shown as mean ± SD. The asterisks (*) indicate significant differences from the mean of 0 h, as analyzed by Student’s t-test (P < 0.05). (G) MACS-purified common carp thrombocytes with surface-bound (upper) or ingested (lower) GFP-expressing E. coli. Colocalization of lysosomal organelles is observed around ingested bacteria (arrowhead). LT, acidic organelles visualized by LysoTracker® Red; E. coli, bacteria; DAPI, nuclei stained with DAPI. Original magnification, ×400. Data are representative of three independent experiments.