T. cruzi trypomastigotes wound mammalian cells and trigger the Ca²⁺-dependent plasma membrane repair response mediated by lysosomal exocytosis. (A) HeLa cells were incubated with trypomastigotes in the presence (membrane repair condition) or absence (nonrepair condition) of Ca²⁺ for 40 min, fixed, and stained for microscopic quantification of intracellular parasites. The data correspond to the mean of triplicates ± SD. ***, P < 0.0001, Student’s t test. (B) Time-lapse live images of cells transduced with Lamp1-RFP and incubated with trypomastigotes in the presence of Ca²⁺. The images show a single confocal plane of cells expressing Lamp1-RFP (green) during interaction with trypomastigotes. The first time point (0 min) was acquired after 20 min incubation of cells with 10⁸ trypomastigotes. Bars, 10 µm. (Video1.) (C) Cell permeabilization and PI (red) influx in response to trypomastigotes in the absence (nonrepair condition) or presence (repair condition) of Ca²⁺. Live cells were imaged by phase contrast and epifluorescence 40 min after trypomastigote addition. Bars, 20 µm. (D) Quantification of the percentage of PI-positive cells (in Ca²⁺ free medium) in comparison with the percentage of cells infected with trypomastigotes (in Ca²⁺-containing medium). Trypomastigotes were allowed to invade cells for the indicated time periods and fixed coverslips were examined to determine the percentage of infected cells or PI-positive cells. Error bars show mean of triplicates ± SD. (E) Fixed cells after incubation with noninfective epimastigotes (Epi) or trypomastigotes (Trypo) in Ca²⁺-free medium in the presence of PI (red). Cell nuclei were stained with DAPI (blue). Bars, 20 µm. These results are representative of three independent experiments.