An Improved Method to Enrich Large Extracellular Vesicles Derived from through Differential Centrifugation.

is a flagellated unicellular protozoan that colonizes the small intestine, causing the diarrheal disease called giardiasis. The production of extracellular vesicles (EVs) by and the role of these EVs in the parasite's interaction with the host have been described. According to biogenesis, EVs are grouped mainly into large (microvesicles-derived from the plasma membrane) and small (exosomes-derived from multivesicular bodies). Populations of EVs are heterogeneous, and improved methods to separate and study them are needed to understand their roles in cell physiology and pathologies. This work aimed to enrich the large extracellular vesicles (LEVs) of in order to better understand the roles of these vesicles in the interaction of the parasite with the host. To achieve the enrichment of the LEVs, we have modified our previously described method and compared it by protein dosage and using Nano tracking analysis. vesiculation was induced by incubation in a TYI-S-33 medium without serum, to which 1 mM of CaCl was added at 37 °C for 1 h. Then, the supernatant was centrifuged at 15,000× for 1 h (15 K 1 h pellet), 15,000× for 4 h (15 K 4 h pellet) and 100,000× for 1.5 h (100 K 1h30 pellet). The pellet (containing EVs) was resuspended in 1× PBS and stored at 4 °C for later analysis. The EVs were quantified based on their protein concentrations using the Pierce BCA assay, and by nanoparticle tracking analysis (NTA), which reports the concentration and size distribution of the particles. The NTA showed that direct ultracentrifugation at 100,000× for 1.5 h and centrifugation at 15,000× for 4 h concentrated more EVs compared to centrifugation at 15,000× for 1 h. Additionally, it revealed that centrifugation at 15,000× 4 h was able to concentrate at the same particle concentration levels as a direct ultracentrifugation at 100,000× for 1.5 h. As for the enrichment of LEVs, the NTA has shown a higher concentration of LEVs in direct ultracentrifugation at 100,000× for 1.5 h, and in centrifugation at 15,000× for 4 h, compared to centrifugation at 15,000× for 1 h. Our results have shown that the most used method at 15,000× for 1 h is not enough to obtain a representative population of large EVs, and we suggest that LEVs released by can be better enriched by direct ultracentrifugation at 100,000× for 1.5 h, or by centrifugation at 15,000× for 4 h.