Differential fluorescence nanoparticle tracking analysis for enumeration of the extracellular vesicle content in mixed particulate solutions.
Antibodies
/ chemistry
Antigens, CD
/ analysis
Cryoelectron Microscopy
Extracellular Vesicles
/ chemistry
Fetal Blood
/ cytology
Filtration
/ methods
Fluoresceins
/ chemistry
Fluorescent Dyes
/ chemistry
Humans
Limit of Detection
Membrane Proteins
/ analysis
Mesenchymal Stem Cells
/ cytology
Nanoparticles
/ analysis
Staining and Labeling
/ methods
Sulfonic Acids
/ chemistry
Ultracentrifugation
/ methods
Journal
Methods (San Diego, Calif.)
ISSN: 1095-9130
Titre abrégé: Methods
Pays: United States
ID NLM: 9426302
Informations de publication
Date de publication:
01 05 2020
01 05 2020
Historique:
received:
25
09
2019
revised:
10
02
2020
accepted:
11
02
2020
pubmed:
23
2
2020
medline:
5
6
2021
entrez:
22
2
2020
Statut:
ppublish
Résumé
A major concern for the extracellular vesicle (EV) field is the current lack of accurate methods for EV quantification. Total protein measurement fails to reliably quantify EVs from serum-containing conditioned media and classical nanoparticle tracking analysis (NTA) allows quantification and size determination of particles, but fails to discriminate between membrane-bounded EVs, lipids and protein aggregates. However, EVs can be fluorescently labelled with non-specific membrane markers or with antibodies specifically recognizing EV surface marker proteins. Fluorescence-based NTA (F-NTA) is thus emerging as a method for counting and phenotyping of EVs. We have validated a differential NTA/F-NTA method using specific antibodies against surface markers in analogy to flow cytometric analyses. EVs from umbilical cord mesenchymal stromal cells (UC-MSCs) were isolated by a combined tangential flow filtration and ultracentrifugation protocol. EV preparations from 2 × 10 All UC-MSC-EV preparations were found positive for typical EV marker proteins and negative for MHC class I. Novel and improved devices that include more sensitive cameras for detection in the fluorescent mode further increase the detection limit. Differential NTA/F-NTA facilitates determination of the percentage of EV marker protein-positive nanoparticles within a mixed particulate solution. The set of markers can be extended to other MSC-EV positive and negative surface proteins in order to establish F-NTA-based profiling as a supporting method for the quantification of EVs.
Sections du résumé
BACKGROUND
A major concern for the extracellular vesicle (EV) field is the current lack of accurate methods for EV quantification. Total protein measurement fails to reliably quantify EVs from serum-containing conditioned media and classical nanoparticle tracking analysis (NTA) allows quantification and size determination of particles, but fails to discriminate between membrane-bounded EVs, lipids and protein aggregates. However, EVs can be fluorescently labelled with non-specific membrane markers or with antibodies specifically recognizing EV surface marker proteins. Fluorescence-based NTA (F-NTA) is thus emerging as a method for counting and phenotyping of EVs. We have validated a differential NTA/F-NTA method using specific antibodies against surface markers in analogy to flow cytometric analyses.
METHODS
EVs from umbilical cord mesenchymal stromal cells (UC-MSCs) were isolated by a combined tangential flow filtration and ultracentrifugation protocol. EV preparations from 2 × 10
RESULTS
All UC-MSC-EV preparations were found positive for typical EV marker proteins and negative for MHC class I. Novel and improved devices that include more sensitive cameras for detection in the fluorescent mode further increase the detection limit.
CONCLUSION
Differential NTA/F-NTA facilitates determination of the percentage of EV marker protein-positive nanoparticles within a mixed particulate solution. The set of markers can be extended to other MSC-EV positive and negative surface proteins in order to establish F-NTA-based profiling as a supporting method for the quantification of EVs.
Identifiants
pubmed: 32081745
pii: S1046-2023(19)30264-6
doi: 10.1016/j.ymeth.2020.02.006
pii:
doi:
Substances chimiques
Antibodies
0
Antigens, CD
0
Fluoresceins
0
Fluorescent Dyes
0
Membrane Proteins
0
Sulfonic Acids
0
alexa fluor 488
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
67-73Informations de copyright
Copyright © 2020 Elsevier Inc. All rights reserved.