Fig. 3

Adhesion assay in F3 and BCEC-1 cells infected by Nc-Spain7 and Nc-Spain1H at 4 hpi. Double immunofluorescence staining was performed, and adhered extracellular tachyzoites were stained with Alexa Fluor® 488 (green) and Alexa Fluor® 594 (red), whereas intracellular tachyzoites were stained with Alexa Fluor® 594 (red). Nuclei were stained with DAPI (blue). Tachyzoites were counted in 10 arbitrarily selected fields, and the percentage of intracellular tachyzoites relative to the number of total adhered tachyzoites at 4 hpi was calculated. Representative images at a magnification of 1000× (a) show the adhesion assay performed in F3 and BCEC-1 cells infected with both isolates. The graph (b) represents the percentage of intracellular tachyzoites of Nc-Spain7 and Nc-Spain1H relative to the total number of intra- and extracellular tachyzoites adhered to F3 and BCEC-1 cells. Each column and error bar represents the mean and the SD of 4 replicates from 2 independent assays. BCEC-1 cells showed a significantly higher percentage of intracellular tachyzoites than F3 cells (P < 0.0001, Chi-square test). The percentage of intracellular tachyzoites for Nc-Spain7 (88%) was significantly higher than for Nc-Spain1H (69%) in F3 (P < 0.0001), whereas the percentage of intracellular tachyzoites of both isolates in BCEC-1 was the same (96%). * represents significant differences between isolates. Scale-bars: a, 40 μm