An Emerging Fluorescence-Based Technique for Quantification and Protein Profiling of Extracellular Vesicles

Robust and well-established techniques for the quantification and characterization of extracellular vesicles (EVs) are a crucial need for the utilization of EVs as potential diagnostic and therapeutic tools. Current bulk analysis techniques such as proteomics and Western blot suffer from low resolution in the detection of small changes in target marker expression levels, exemplified by the heterogeneity of EVs. Microscopy-based techniques can provide valuable information from individual EVs; however, they are time-consuming and statistically less powerful than other techniques. Flow cytometry has been successfully employed for the quantification and characterization of individual EVs within larger populations. However, traditional flow cytometry is not highly suited for the examination of smaller, submicron particles. Here we demonstrate the accurate and precise quantification of nanoparticles such as EVs using the Virus Counter 3100 (VC3100) platform, a fluorescence-based technique that uses the principles of flow cytometry with critical enhancements to enable the effective detection of smaller particles. This approach can detect nanoparticles precisely with no evidence of inaccurate concentration measurement from masking effects associated with traditional nanoparticle tracking analysis (NTA). Fluorescently labeled EVs from different sources were successfully quantified using the VC3100 without a postlabeling washing step. Moreover, protein profiling and characterization of individual EVs were achieved and have been shown to determine the expression level of target protein markers.