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. 2012 Oct 4;6(10):e1804. doi: 10.1371/journal.pntd.0001804

Figure 2. Differential expression of T. cruzi Y82 and Y30 strain surface molecules implicated in host cell invasion.

Figure 2

Live metacyclic trypomastigotes were incubated for 1 h with mAb 3F6 (A) or polyclonal anti-J18 antibodies (B) and processed for flow cytometry analysis, or for observations at the fluorescence microscope along with paraformaldehyde-fixed saponin-permeabilized parasites. In parallel, detergent-solubilized parasite extracts were analysed by immunoblotting. C) HeLa cells were incubated for 1 h with metacyclic forms, fixed and stained with Giemsa, followed by dehydration as described in experimental procedures. The number of intracellular parasites was counted in a total of 250 cells. The values are the means ± SD of eight independent experiments performed in duplicate. Shown on the right are HeLa cells with internalized metacyclic forms (black arrows), which are surrounded by a clear space, and adherent parasites (white arrow). Scale bar = 10 µm. D) HeLa cells were incubated with metacyclic forms for 1 h, washed to remove non internalized parasites, and incubation proceeded for 24 h in DMEM containing 2% fetal calf serum, upon which the cells were fixed and stained as in (C) for parasite counting. The values are the means ± SD of four independent assays performed in duplicate. Shown on the right are HeLa cells with amastigotes (black arrows). Scale bar = 10 µm. E) HeLa cells were incubated with metacyclic forms, untreated or pretreated with mAb 3F6, and the cells were processed as in (C). The values are the means ± SD of three independent experiments performed in duplicate. The difference between the parasites treated with mAb 3F6 and the untreated control was significant (*p<0.005, **p<0.01). F) Metacyclic forms, untreated or pretreated with mAb 3F6, were incubated for 1 h with HeLa cells, non internalized parasites were washed away, and incubation proceeded for 24 h as in (D). The values are the means ± SD of three independent assays performed in duplicate. MAb 3F6 significantly inhibited parasite invasion (*p<0.005, **p<0.01).