Spectral flow cytometry for detecting DNA cargo in malaria parasite–derived extracellular vesicles

Cells across biological kingdoms release extracellular vesicles (EVs) as a means of communication with other cells, be their friends or foes. This is indeed true for the intracellular malaria parasite Plasmodium falciparum (Pf), which utilizes EVs to transport bioactive molecules to various human host systems. Yet, the study of this mode of communication in malaria research is currently constrained due to limitations in high-resolution tools and the absence of commercial antibodies. Here, we demonstrate the power of an advanced spectral flow cytometry approach to robustly detect secreted EVs, isolated from Pf-infected red blood cells. By labeling both EV membrane lipids and the DNA cargo within (non-antibody staining approach), we were able to detect a subpopulation of parasitic-derived EVs enriched in DNA. Furthermore, we could quantitatively measure the DNA-carrying EVs isolated from two distinct blood stages of the parasite: rings and trophozoites. Our findings showcase the potential of spectral flow cytometry to monitor dynamic changes in nucleic acid cargo within pathogenic EVs.