Systematic methodological evaluation of a multiplex bead-based flow cytometry assay for detection of extracellular vesicle surface signatures

• Published in: Journal of extracellular vesicles Vol. 7; p. 269
• Main Authors: Wiklander, Oscar P B, Bostancioglu, Beklem, Felldin, Ulrika, Zickler, Antje, Murke, Florian, Welsh, Joshua A, Evertsson, Björn, Liang, Xiu-Ming, Corso, Giulia, Gustafsson, Manuela, Mohammad, Dara, Wiek, Constanze, Hanenberg, Helmut, Bremer, Michel, Gupta, Dhanu, Björnstedt, Mikael, Jones, Jennifer, Giebel, Bernd, Nordin, Joel Z, El-Andaloussi, Samir, Görgens, André
• Format: Journal Article
• Language: English
• Published: Abingdon John Wiley & Sons, Inc 01.01.2018
• Subjects: Body fluids; Cell culture; Cell lines; Exosomes; Flow cytometry; Medical research; Progenitor cells; Surface markers; Ultracentrifugation
• ISSN: 2001-3078

Background: Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids and can be roughly classified as exosomes and microvesicles. Nowadays, it is commonly accepted in the field that there is a much higher degree ofEV heterogeneitywithin these two subgroups than previously thought. Moreover, surface marker signatures of EVs are likely to be dependent on the cell type source and other multiple parameters. To date, no specific markers to even discriminate exosomes from microvesicles have been identified yet, and only few EV surface markers have been related to specific cell sources. Methods: In the last few years, improved flow-cytometric assays have been developed. However, most assays are limited to dedicated instruments and require extensive operator expertise. In this study, we systematically evaluated the use of a multiplex bead-based assay which can be used with most standard flow cytometers for detection of human EV surface signatures. Results: First, we assessed assay variability, sample stability over time, dynamic range and the limitations of this assay in terms of EV input quantity and EV concentration on different instruments. Next, we compared how EV sample quality affects assay results and found that this assay was fit for purpose in detecting, quantifying and comparing EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different isolation methods (differential ultracentrifugation, tangential flow filtration and size-exclusion chromatography), as well as biological fluids. Furthermore, we explored the use and limitations of this assay to assess heterogeneities in EV surface signatures by combining different sets of detection antibodies in EV samples derived from different immortalized cell lines. Finally, we demonstrate differential detection of EV surface markers in supernatants of primary haematopoietic progenitor cell subsets after short-term culture in low cell doses of 25,000 cells/well. Summary/Conclusion: Taken together, we show that this multiplex bead-based flow cytometric assay allows robust, sensitive and reproducible detection and quantification of EV surface marker expression in various sample types, which will be extremely valuable for many researchers working in the EV field.