Mutant ratio calculation by microscopy

About 10 μl of infected blood were collected in 200 μl of PBS, and 2 μl each of heparin solution and Hoechst33342 (1 mM) were added. The sample was then incubated at 37 °C for 5 min. After incubation, the sample was harvested at 500 × g for 5 min, and resuspended in 25–30 μl of RPMI1640. The suspension was placed on a glass slide, and the number of eGFP-positive parasites stained with Hoechst33342 was determined using fluorescent microscopy. The parasites were distinguished from white blood cells by fluorescence intensity and morphology of nuclei stained with Hoechst. At least 1,000 Hoechst-positive parasites were counted in several fields under 400× magnification. The mutant ratio was calculated by dividing the number of eGFP-positive parasites by the number of Hoechst-positive parasites. In experiments where transfection was performed with GFP-positive parasites, the mutant ratio was calculated by dividing the number of GFP-negative parasites by the number of Hoechst-positive parasites.