2.6. Parasite Plaque Assay

For the plaque assay, HFF monolayers in a 12-well cell plate (Thermo Fisher Scientific) were infected with freshly egressed RHΔzfp strains and WT strain (approximately 500 tachyzoites per well). The plates were incubated at 37 °C, 5% CO₂ for 7–9 days without any interference. Then, the culture medium was removed, and the infected monolayers were fixed with 4% PFA. For plaque visualization, the sample was stained with 0.2% crystal violet for a duration of 20 min. The size and number of plaques per well were analyzed using the ImageJ 1.46 software. Three biology replicates were performed for each sample.