Two Complementary Strategies to Quantitate Extracellular Vesicle Uptake Using Bioluminescence and Non-Lipidic Dyes

The study of the molecular mechanisms controlling extracellular vesicle uptake by a target cell is an aspect of great interest within the EV community due to EV relevance in intercellular communication for tissue homeostasis or different disease progressions such as cancer or Alzheimer's. Since the EV field is relatively young, standardization of techniques for even basic aspects such as their isolation and characterization is still under development and debate. So it is for the study of EV uptake, where the currently most used strategies have critical limitations. Newly designed techniques should try to discern the uptake events from the surface EV binding or to improve the sensitivity and fidelity of the assays. Here, we describe two different complementary methods to measure and quantify EV uptake that we believe, help to overcome certain limitations of the currently used techniques. One is based on a mEGFP-Tspn-Rluc construct, to sort these two reporters into EVs. The use of bioluminescence signal to measure EV uptake allows for a better sensitivity, discerns EV binding from uptake, and allows kinetics measurement in alive cells, being compatible with a high-throughput screen format. The second one is a flow cytometry assay based in EV staining with a maleimide conjugated with a fluorophore, a chemical compound that covalently binds to proteins within sulfhydryl residues, being a good alternative to lipidic dyes and compatible with flow cytometry sorting of cell populations that have captured the labeled EVs.