Isolation of erythrocytes infected with viable early stages of Plasmodium falciparum by flow cytometry.

ABSTRACT

The erythrocytic life cycle of Plasmodium falciparum is highly associated with severe clinical symptoms of malaria that causes hundreds of thousands of death each year. The parasite develops within human erythrocytes leading to the disruption of the infected red blood cell (iRBC) prior to the start of a new cycle of erythrocyte infection. Emerging mechanisms of resistance against antimalarial drugs require improved knowledge about parasite's blood stages to facilitate new alternative antimalarial strategies. For the analysis of young blood stages of Plasmodium at the molecular level, the isolation of ring stages is essential. However, early stages can hardly be separated from both, late stages and non-infected red blood cells using conventional methods. Here, iRBCs were stained with the DNA-binding dyes Vybrant® DyeCycle™ Violet and SYBR® Green I. Subsequently, cells were subjected to flow-cytometric analysis. This enabled the discrimination of early stage iRBCs as well as late-stage iRBCs from non-infected erythrocytes and the properties of the used dyes were evaluated. Moreover, early stage iRBCs were isolated with high purity (>98%) by FACS. Subsequently, development of sorted early stages of the parasite was monitored over time and compared with control cultures. The described flow cytometry method, based on staining with Vybrant DyeCycle Violet, allows the isolation of viable ring stages of the malarial agent P. falciparum, and thereby provides the basis for new, broad-range molecular investigations of the parasite.