Data_Paper_De_Putter

Description

Abstract

Exosomes are nanosized extracellular vesicles pivotal in numerous physiological and pathological processes, such as immune responses, viral pathogenesis, pregnancy, cardiovascular diseases, and cancer progression. Their capacity to influence complex intracellular pathways highlights their therapeutic potential in addressing various conditions, including neurodegenerative disorders and cancer.  A novel capillary isotachophoresis (cITP) method was developed for the electrokinetic characterization of pre-isolated exosomes. Over 14 distinct peaks could be resolved at near-baseline resolution using a novel mixture of spacer ions and laser-induced fluorescence (LIF) detection. Exosomes were effectively separated from the unbound CFDA-SE amine-reactive fluorescent stain used to detect them and from residual contaminants. Using an OV1701-OH deactivated capillary system, we achieved a reproducible separation of exosomes derived from plasma and two different cellular origins. The identity of the peaks shown in the electropherograms was validated via various methods, including incubation with specific antibodies, or spiking of putative contaminants, such as the fluorescent dye for tracking the exosomes, proteins, and lipoproteins. This report thus provides a detailed proof-of-concept for using cITP-LIF for exosome isolation, subtype fractionation, and profiling.