L. pneumophila–containing vacuoles are devoid of PS. (A) RAW macrophages transfected with sec61α-GFP were infected with L. pneumophila for 2 h, washed, and further incubated for 2–4 h. Cells were fixed and permeabilized with cold methanol, and the internalized bacteria were stained with an anti–L. pneumophila antibody followed by a Cy3-conjugated secondary antibody. (B and C) RAW macrophages transfected with the PS biosensor mRFP-Lact-C2 were infected with live (B) or were allowed to phagocytose PFA-treated (C) L. pneumophila expressing GFP-flaA for 2 h. The RAW macrophages were washed and further incubated for 2–4 h. Insets in C show a magnification of the boxed areas showing a Lact-C2–positive L. pneumophila–containing vacuole. Images in A–C are representative of 30 cells from two similar experiments. (D) Quantification of the percentage of vacuoles containing either live or PFA-killed L. pneumophila that bound Lact-C2. Data are means ± SEM quantified from >30 phagosomes. Bars, 2 µm.