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Single extracellular vesicle analysis performed by imaging flow cytometry in contrast to NTA rigorously assesses the accuracy of urinary extracellular vesicle preparation techniques
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Single extracellular vesicle analysis performed by imaging flow cytometry in contrast to NTA rigorously assesses the accuracy of urinary extracellular vesicle preparation techniques
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Single extracellular vesicle analysis performed by imaging flow cytometry in contrast to NTA rigorously assesses the accuracy of urinary extracellular vesicle preparation techniques
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Paper
Single extracellular vesicle analysis performed by imaging flow cytometry in contrast to NTA rigorously assesses the accuracy of urinary extracellular vesicle preparation techniques
2021
Overview
Extracellular vesicles (EVs) from several body fluids, including urine, appear as promising biomarkers. Within the last decade, numerous groups have compared the efficacy of EV preparation protocols. Frequently, the efficacy of EV preparation methods is judged by the recovery of particles as estimated by conventional nanoparticle tracking analysis (NTA) or other particle quantification devices. Here, at the example of different urinary EV (uEV) preparation methods, we determined the particle yield in obtained samples with conventional NTA, analyzed their EV content by imaging flow cytometry (IFCM) and quantified the intensity of TSG101 and the contaminant protein uromodulin (UMOD) in Western blots. Our results demonstrate a correlation among CD9-positive objects detected by IFCM and TSG101 Western blot intensities, while particle numbers as determined by NTA correlated with the amount of UMOD. Consequently, our results question the reliability of conventional NTA analyses for identifying the optimal EV preparation method. Here, in our method comparison, a combination of size exclusion chromatography followed by ultra-filtration showed the highest CD9-positive object and TSG101 protein recovery, and in relation to the number of CD9-positive objects, the lowest amount of UMOD contamination. Competing Interest Statement B.G. is a scientific advisory board (SAB) member of Evox Therapeutics and Innovex Therapeutics. Furthermore, he is founding director of Exosla. All other authors have declared that no competing interests exist.
Publisher
Cold Spring Harbor Laboratory Press,Cold Spring Harbor Laboratory
Subject
