Assessment of the presence of PS in C. trachomatis inclusion vacuoles. (A) RAW macrophages transfected with GFP-Lact-C2 were infected with C. trachomatis elementary bodies for 6 h. Cells were fixed and permeabilized with cold methanol, and the internalized bacteria were stained with polyclonal anti–C. trachomatis antibodies followed by Cy3-conjugated secondary antibody. Insets highlight the typical vacuole marked by the boxed regions. (B) RAW macrophages transfected with mRFP-Lact-C2 were infected with C. trachomatis elementary bodies for 18 h. Cells were incubated with the DNA stain Draq5 to identify the C. trachomatis invasion vacuoles (x). N indicates the location of the macrophage nucleus. Images are representative of 30 cells from three similar experiments. (C) Quantification of the percentage of C. trachomatis inclusion vacuoles that bound Lact-C2 at 6 and 18 h. Data are means ± SEM quantified from at least 50 phagosomes from two experiments. Bars, 2 µm.