TY - JOUR
T1 - An imaging flow cytometry-based methodology for the analysis of single extracellular vesicles in unprocessed human plasma
AU - Woud, Wouter W.
AU - van der Pol, Edwin
AU - Mul, Erik
AU - Hoogduijn, Martin J.
AU - Baan, Carla C.
AU - Boer, Karin
AU - Merino, Ana
N1 - Funding Information: The authors would like to thank Peter Rhein (Luminex) for his support and discussions throughout the project. Publisher Copyright: © 2022, The Author(s).
PY - 2022/12/1
Y1 - 2022/12/1
N2 - Extracellular vesicles (EVs) are tissue-specific particles released by cells containing valuable diagnostic information in the form of various biomolecules. To rule out selection bias or introduction of artefacts caused by EV isolation techniques, we present a clinically feasible, imaging flow cytometry (IFCM)–based methodology to phenotype and determine the concentration of EVs with a diameter ≤400 nm in human platelet-poor plasma (PPP) without prior isolation of EVs. Instrument calibration (both size and fluorescence) were performed with commercial polystyrene beads. Detergent treatment of EVs was performed to discriminate true vesicular events from artefacts. Using a combination of markers (CFSE & Tetraspanins, or CD9 & CD31) we found that >90% of double-positive fluorescent events represented single EVs. Through this work, we provide a framework that will allow the application of IFCM for EV analysis in peripheral blood plasma in a plethora of experimental and potentially diagnostic settings. Additionally, this direct approach for EV analysis will enable researchers to explore corners of EVs as cellular messengers in healthy and pathological conditions.
AB - Extracellular vesicles (EVs) are tissue-specific particles released by cells containing valuable diagnostic information in the form of various biomolecules. To rule out selection bias or introduction of artefacts caused by EV isolation techniques, we present a clinically feasible, imaging flow cytometry (IFCM)–based methodology to phenotype and determine the concentration of EVs with a diameter ≤400 nm in human platelet-poor plasma (PPP) without prior isolation of EVs. Instrument calibration (both size and fluorescence) were performed with commercial polystyrene beads. Detergent treatment of EVs was performed to discriminate true vesicular events from artefacts. Using a combination of markers (CFSE & Tetraspanins, or CD9 & CD31) we found that >90% of double-positive fluorescent events represented single EVs. Through this work, we provide a framework that will allow the application of IFCM for EV analysis in peripheral blood plasma in a plethora of experimental and potentially diagnostic settings. Additionally, this direct approach for EV analysis will enable researchers to explore corners of EVs as cellular messengers in healthy and pathological conditions.
UR - http://www.scopus.com/inward/record.url?scp=85132970990&partnerID=8YFLogxK
U2 - https://doi.org/10.1038/s42003-022-03569-5
DO - https://doi.org/10.1038/s42003-022-03569-5
M3 - Article
C2 - 35768629
SN - 2399-3642
VL - 5
JO - Communications Biology
JF - Communications Biology
IS - 1
M1 - 633
ER -