Establishment of Hoechst and GFP detection by cytometry. Upper panel, Hoechst staining of infected red blood cells (iRBCs) at day 2 of the assay demonstrates that the majority of parasites have 1 nucleus (ie, rings and gametocytes [R + G]), whereas a subset is multinucleated (trophozoites and schizonts [T + S]). Both fresh uninfected red blood cells (uRBCs; from 4°C) and uRBCs incubated along for the assay period and stained with Hoechst were used as a negative control for gating. Lower panel, iRBCs at day 2 of the assay show GFP–positive population. This population is significantly higher than the background signal represented by autofluorescence in uRBC control samples that were either fresh or were incubated for the entire assay period.