FIG. 3.

Difference in the phagocytosis of L. monocytogenes (LM) between WT and MyD88^−/− macrophages. (A) WT and MyD88^−/− macrophages were infected with L. monocytogenes at an MOI of 20 for 1 h. After being washed, cells were treated with gentamicin for 30 min and the number of phagocytosed bacteria was determined. Data are expressed as the means ± standard deviations (SD) for results of triplicate cultures. Similar results were obtained in three independent experiments. *, P < 0.05. (B) WT and MyD88^−/− macrophages were infected with CFSE-labeled L. monocytogenes at an MOI of 50 for 1 h. The numbers of phagocytosed L. monocytogenes and adherent L. monocytogenes cells were evaluated after immunostaining. Data are expressed as the means ± SD for results of triplicate cultures. Similar results were obtained in two independent experiments. *, P < 0.05. (C) WT and MyD88^−/− macrophages were incubated with polystyrene beads for 2 h. After being washed, cells were fixed and the number of internalized beads was counted. (D) WT and MyD88^−/− macrophages were infected with CFSE-labeled L. monocytogenes at an MOI of 50 for 1 h and treated similarly to those shown in Fig. 2E. The viability of intracellular bacteria was calculated as a percentage. (E) WT, TLR2^−/−, and MyD88^−/− macrophages were stained with FITC-conjugated anti-TLR2 MAb (IgG2b) or control IgG2b, and the level of TLR2 expression was analyzed by FACS. The histogram with the bold line shows the level of TLR2 expression on the three types of macrophages. The hatched area represents the basal fluorescent intensity in cells treated with control Ab. (F) Whole PEC and adherent PEC obtained from WT (red), TLR2^−/− (blue), and MyD88^−/− (green) mice were stained with PE-conjugated anti-F4/80 MAb (IgG2a). The expression of F4/80 was analyzed by FACS. The hatched area represents the basal fluorescent intensity in cells treated with control IgG2a.