Visualizing Synchronized Parasite Egress

Egress was visualized by differential inference contrast (DIC) light microscopy as described previously (26 (link)) using a Nikon Eclipse Ni microscope with a Hamamatsu C11440 digital camera. The PKG (cGMP-dependent protein kinase G) inhibitor compound 2 (4-[7-[(dimethylamino)methyl]-2-(4-fluorphenyl)imidazo[1,2-α]pyridine-3-yl]pyrimidin-2-amine) was used to tightly synchronize parasites prior to egress, as described previously (26 (link)). To visualize egress in both DMSO- and RAP-treated cultures concurrently, one culture was stained with 1 µg/ml Hoechst stain (Thermo) for 5 min before washing and pooling the cultures and imaging, as previously described (30 (link)). The percentage of parasites undergoing egress and the time to egress were quantified from nine videos, and the statistical significance of any differences between the DMSO- and RAP-treated parasites was calculated using paired t tests.