Fig. 1

Use of a fluorochrome-based flow cytometer assay for the assessment of Plasmodium falciparum development. a Schematic diagram of the process to assess Plasmodium development in a culture using fluorescence dyes and flow cytometry. In the left panel, sexual and asexual development of P. falciparum are shown. Plasmodium parasites asexually develop through the following stages in a cyclical manner in culture: ring-shaped trophozoites, trophozoites, and schizonts. Upon rupture of a schizont, merozoites egress from the host cells and subsequently invade new erythrocytes, initiating the next developmental cycle. Some ring-shaped trophozoites undergo sexual development, resulting in gametocytes. The middle panel shows that to detect Plasmodium-infected erythrocytes, parasite nucleic acids of all intraerythrocytic stages could be stained with a number of fluorescent dyes. Then, the cells are taken into a flow cytometer, by which each cell is individually treated in a flowing fluid. The cells are exposed to light of different wavelengths generated from the laser light source. Nucleic acid-binding fluorochromes can be excited by light, resulting in fluorescence emission. In addition, cell size and intracellular content also cause scatter of the exposed light in the forward and side directions. A flow cytometer obtains a fluorescence signal from the emitted fluorescence and the forward- and side-scattered light. The fluorescence signal is displayed based on the fluorescence intensity. The emitted fluorescence is shown as a yellow Y-axis, while the forward scatter (FSC) or side scatter (SSC) is shown as a green X-axis (right panel). In the right panel, an example of data analysis post flow cytometry is illustrated. To determine the threshold to distinguish cells with emitted fluorescence, an unstained cell sample (− fluorochrome) is included. For the stained cells (+ fluorochrome), a mixture of cells is clearly discriminated based on resolving the emitted fluorescence intensity into three categories: high, intermediate and low. Here, when the fluorescence intensity is proportional to the nucleic acid content, each developmental stage of Plasmodium is identified: ring-form and early trophozoites have the lowest fluorescence intensity, late schizonts have intermediate fluorescence intensity, and mature schizonts have the highest fluorescence intensity. b An application of fluorochrome-based flow cytometric analysis for drug susceptibility testing. Generally, a synchronous culture of ring-formed P. falciparum is incubated with an anti-malarial drug (here referred to as X) and then stained with fluorochrome. Without drug X (control), the ring-form stage of P. falciparum develops into trophozoites and schizonts. In contrast, the drug-treated Plasmodium parasites develop at slower speeds or arrest between the ring form and the mature trophozoite, resulting in a lower percentage of trophozoites. c Examples of analysed data displayed in two formats. First, a dose-response curve can be generated regardless of the Plasmodium stage, but only parasitaemia, the percentage of cells having a fluorescence signal, is calculated. Second, according to the developmental stage, the bar graph can additionally inform which stage is affected by the drug. Based on the displayed data, drug X likely inhibits the development of the ring form into mature trophozoites