Single Extracellular Vesicle Analysis Performed by Imaging Flow Cytometry and Nanoparticle Tracking Analysis Evaluate the Accuracy of Urinary Extracellular Vesicle Preparation Techniques Differently

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 mic...

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Autores principales:	Marvin Droste, Tobias Tertel, Stefanie Jeruschke, Robin Dittrich, Evangelia Kontopoulou, Bernd Walkenfort, Verena Börger, Peter F. Hoyer, Anja K. Büscher, Basant K. Thakur, Bernd Giebel
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	imaging flow cytometry extracellular vesicles urine extracellular vesicle isolation methods exosomes nanoparticle tracking analysis Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/804edd60f6b8494487c34634acd43f45
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spelling	oai:doaj.org-article:804edd60f6b8494487c34634acd43f452021-11-25T17:56:38ZSingle Extracellular Vesicle Analysis Performed by Imaging Flow Cytometry and Nanoparticle Tracking Analysis Evaluate the Accuracy of Urinary Extracellular Vesicle Preparation Techniques Differently10.3390/ijms2222124361422-00671661-6596https://doaj.org/article/804edd60f6b8494487c34634acd43f452021-11-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/22/12436https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067Small 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<sup>+</sup> sEV population, which exceeds CD63<sup>+</sup> and CD81<sup>+</sup> sEV populations. Furthermore, we demonstrated that the storage temperature of urine samples negatively affects the recovery of CD9<sup>+</sup> sEVs. Although overall reduced, the highest CD9<sup>+</sup> sEV recovery was obtained from urine samples stored at −80 °C and the lowest from those stored at −20 °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.Marvin DrosteTobias TertelStefanie JeruschkeRobin DittrichEvangelia KontopoulouBernd WalkenfortVerena BörgerPeter F. HoyerAnja K. BüscherBasant K. ThakurBernd GiebelMDPI AGarticleimaging flow cytometryextracellular vesiclesurineextracellular vesicle isolation methodsexosomesnanoparticle tracking analysisBiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 12436, p 12436 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	imaging flow cytometry extracellular vesicles urine extracellular vesicle isolation methods exosomes nanoparticle tracking analysis Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	imaging flow cytometry extracellular vesicles urine extracellular vesicle isolation methods exosomes nanoparticle tracking analysis Biology (General) QH301-705.5 Chemistry QD1-999 Marvin Droste Tobias Tertel Stefanie Jeruschke Robin Dittrich Evangelia Kontopoulou Bernd Walkenfort Verena Börger Peter F. Hoyer Anja K. Büscher Basant K. Thakur Bernd Giebel Single Extracellular Vesicle Analysis Performed by Imaging Flow Cytometry and Nanoparticle Tracking Analysis Evaluate the Accuracy of Urinary Extracellular Vesicle Preparation Techniques Differently
description	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<sup>+</sup> sEV population, which exceeds CD63<sup>+</sup> and CD81<sup>+</sup> sEV populations. Furthermore, we demonstrated that the storage temperature of urine samples negatively affects the recovery of CD9<sup>+</sup> sEVs. Although overall reduced, the highest CD9<sup>+</sup> sEV recovery was obtained from urine samples stored at −80 °C and the lowest from those stored at −20 °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.
format	article
author	Marvin Droste Tobias Tertel Stefanie Jeruschke Robin Dittrich Evangelia Kontopoulou Bernd Walkenfort Verena Börger Peter F. Hoyer Anja K. Büscher Basant K. Thakur Bernd Giebel
author_facet	Marvin Droste Tobias Tertel Stefanie Jeruschke Robin Dittrich Evangelia Kontopoulou Bernd Walkenfort Verena Börger Peter F. Hoyer Anja K. Büscher Basant K. Thakur Bernd Giebel
author_sort	Marvin Droste
title	Single Extracellular Vesicle Analysis Performed by Imaging Flow Cytometry and Nanoparticle Tracking Analysis Evaluate the Accuracy of Urinary Extracellular Vesicle Preparation Techniques Differently
title_short	Single Extracellular Vesicle Analysis Performed by Imaging Flow Cytometry and Nanoparticle Tracking Analysis Evaluate the Accuracy of Urinary Extracellular Vesicle Preparation Techniques Differently
title_full	Single Extracellular Vesicle Analysis Performed by Imaging Flow Cytometry and Nanoparticle Tracking Analysis Evaluate the Accuracy of Urinary Extracellular Vesicle Preparation Techniques Differently
title_fullStr	Single Extracellular Vesicle Analysis Performed by Imaging Flow Cytometry and Nanoparticle Tracking Analysis Evaluate the Accuracy of Urinary Extracellular Vesicle Preparation Techniques Differently
title_full_unstemmed	Single Extracellular Vesicle Analysis Performed by Imaging Flow Cytometry and Nanoparticle Tracking Analysis Evaluate the Accuracy of Urinary Extracellular Vesicle Preparation Techniques Differently
title_sort	single extracellular vesicle analysis performed by imaging flow cytometry and nanoparticle tracking analysis evaluate the accuracy of urinary extracellular vesicle preparation techniques differently
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/804edd60f6b8494487c34634acd43f45
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Single Extracellular Vesicle Analysis Performed by Imaging Flow Cytometry and Nanoparticle Tracking Analysis Evaluate the Accuracy of Urinary Extracellular Vesicle Preparation Techniques Differently

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