Vacuoles containing recently internalized parasites are enriched in ceramide while gradually acquiring Lamp1. (A) Single optical section of a HeLa cell expressing Lamp1-RFP (green) infected with trypomastigotes for 15 min, fixed, permeabilized, and stained with anti-ceramide antibodies (red). Trypomastigotes (arrows) can be observed in vacuoles enriched in ceramide (red), of which one has already fused with Lamp1-RFP–containing lysosomes (green). The arrowhead indicates a Lamp1-positive parasite vacuole that is negative for ceramide. Bar, 10 µm. (B) Quantification of intracellular parasites found in ceramide or Lamp1-enriched vacuoles over time. After 10 or 20 min of exposure to trypomastigotes, cells were washed and either fixed or incubated for an additional 30 min before fixation (50-min time point). Cells were then permeabilized, stained with antibodies to ceramide (red) or Lamp1 (green), and confocal Z series were obtained in 15 fields for each condition, followed by quantification of parasites associated with ceramide and Lamp1. The data represents mean ± SD of the percentage of positive parasites per field (n = 15). (C) Representative images (single optical sections) of each time point in B. At 10 and 20 min, protruding parasites were often observed in ceramide-enriched vacuoles (arrows). Ceramide, red; Lamp1, green; DAPI, blue. The arrowheads indicate a Lamp1-positive parasite vacuole that is negative for ceramide. Bars, 5 µm. These results are representative of three independent experiments.