Flow cytometric procedures for deep characterization of nanoparticles

In recent years, there has been a notable increasing interest surrounding the identification and quantification of nano-sized particles, including extracellular vesicles (EVs) and viruses. The challenge posed by the nano-sized dimension of these particles makes precise examination a significant undertaking. Among the different techniques for the accurate study of EVs, flow cytometry stands out as the ideal method. It is characterized by high sensitivity, low time consumption, non-destructive sampling, and high throughput. In this article, we propose the optimization of flow cytometry procedures to identify, quantify, and purify EVs and virus-like particles. The protocol aims to reduce artefacts and errors in nano-sized particles counting, overall caused by the swarming effect. Different threshold strategies were compared to ensure result specificity. Additionally, the critical parameters to consider when using conventional flow cytometry outside of the common experimental context of use have also been identified. Finally, fluorescent-EVs sorting protocol was also developed with highly reliable results using a conventional cell sorter.