As researchers push to gain further insight into extracellular vesicles (EVs), the characterization of EVs remain a growing topic. EVs extracted from body fluids are typically accompanied by non-EV components creating a diverse and heterogeneous system that poses significant challenges for analytical characterisation. The tetraspanins are integral trans-membrane proteins intricately involved in various aspects of extracellular vesicle biology. They are often considered as the most accessible biomarkers for research due to their high abundance on the surface of many EV subsets. They are often utilised in EV profiling and as specific exosome markers, hence the ability to detect these biomarkers holds considerable importance in EV research. The protocols for carrying out such a measurement can be challenging because there are multiple variables that are required to be investigated and optimised.
In this study, we have conducted comprehensive labelling methods on urine-derived EVs employing three different labelling strategies. Firstly, we successfully recognised membrane-labeled EVs with CMO™ and ExoGlow™ in order to recognise EVs, without affecting their original size and concentration. Secondly, the presence of all three tetraspanin biomarkers (CD9, CD63 and CD81) was measured for decoding EV´s personality. Last but not least, RNA was successfully labeled with Quant-iT RiboGreen™ for discovering EVs secrets without destroying the EVs.
A range of fluorescent dyes and fluorescent antibodies were used in this study including: CellMask™ Orange (CMO), ExoGlow™, Alexa Fluor™ 488 anti-human CD9, CD63, CD81 Antibody, Qant-iT™RiboGreen.
Here we demonstrate that while the EXODUS system addresses key challenges in exosome isolation by providing a reliable, scalable, and reproducible solution, the new NanoSight Pro NTA instrument with a specialised fluorescence mode achieved rapid characterisation to validate and identify EVs and their subpopulations which is crucial for advancing EV research.