Gating for detection of depolarizing parasitized red blood cells in a <i>Plasmodium falciparum</i> culture.

<p>Flow cytometric analysis of an uninfected and a synchronized <i>P. falciparum</i> (3D7) infected culture (1.5% parasitemia) after 24 hours of incubation, stained with SYBR green I. Plots of forward vs. side scatter for the uninfected and infected cultures appear in Figures A and B; corresponding plots of side scatter vs. depolarized side scatter appear in Figures C and D. The gates in Figures C and D identify the depolarizing events. Figures E and F (see text) illustrate gates defining SYBR green-positive parasitized cells. The blue dots on Figure F represent the depolarizing events. Staining with the red blood cell surface marker (CD235) shows that 99.5% of events in a stained sample (red line) exhibit fluorescence above the highest level measured in an unstained control (black line), indicating that the detected events are red blood cells (G). In the SYBR green I histogram (H) of the infected culture, the overall population (black line) shows a distinct peak with a high fluorescent intensity in the third decade. This peak corresponds mainly to the gated population of depolarizing events (pink line). Because SYBR green I intensity correlates with DNA content and thus with parasite level of maturation, the depolarizing population (pink line) consists mainly (79.4%) of mature parasites. The highly red- and green-fluorescent events visible outside the SYBR green gate just to the right of its apex represent contaminating white blood cells among the donor red cells.