Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 22, Issue 22, Pages -Publisher
MDPI
DOI: 10.3390/ijms222212436
Keywords
imaging flow cytometry; extracellular vesicles; urine; extracellular vesicle isolation methods; exosomes; nanoparticle tracking analysis
Funding
- Else Kroener-Fresenius-Stiftung
Ask authors/readers for more resources
Small extracellular vesicles isolated from urine (uEVs) are considered potential biomarkers, with different preparation strategies available. Analysis using nanoparticle tracking analysis (NTA), Western blot, and imaging flow cytometry (IFCM) showed that urine mainly contains CD9(+) sEV population, storage temperature affects CD9(+) sEV recovery, and challenges the reliability of traditional NTA for evaluating EV preparation methods.
Small extracellular vesicles isolated from urine (uEVs) are increasingly recognized as potential biomarkers. Meanwhile, different uEV preparation strategies exist. Conventionally, the performance of EV preparation methods is evaluated by single particle quantification, Western blot, and electron microscopy. Recently, we introduced imaging flow cytometry (IFCM) as a next-generation single EV analysis technology. Here, we analyzed uEV samples obtained with different preparation procedures using nanoparticle tracking analysis (NTA), semiquantitative Western blot, and IFCM. IFCM analyses demonstrated that urine contains a predominant CD9(+) sEV population, which exceeds CD63(+) and CD81(+) sEV populations. Furthermore, we demonstrated that the storage temperature of urine samples negatively affects the recovery of CD9(+) sEVs. Although overall reduced, the highest CD9(+) sEV recovery was obtained from urine samples stored at -80 & DEG;C and the lowest from those stored at -20 & DEG;C. Upon comparing the yield of the different uEV preparations, incongruencies between NTA and IFCM data became apparent. Results obtained by both NTA and IFCM were consistent with Western blot analyses for EV marker proteins; however, NTA results correlated with the amount of the impurity marker uromodulin. Despite demonstrating that the combination of ultrafiltration and size exclusion chromatography appears as a reliable uEV preparation technique, our data challenge the soundness of traditional NTA for the evaluation of different EV preparation methods.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available