Isolation of Small Extracellular Vesicles from Human Sera
Abstract
:1. Introduction
2. Results
2.1. Nanoparticle Tracking Analysis of Serum-Derived sEV Purified by DUC or Exo-spin™
2.2. Analysis of the Abundance of Exosomal Markers by Western Blotting and ELISA
2.3. Ultrastructure of sEV Preparations Isolated by Exo-spin™ and DUC and Analysed by Transmission Electron Microscopy (TEM)
2.4. Co-Purified Lipoproteins Account for the Excess Particles in Exo-spin™ Preparations
2.5. MACSPlex Analysis of Membrane Proteins on sEV Purified by DUC and Exo-spin™
3. Discussion
4. Materials and Methods
4.1. Blood Samples
4.2. Isolation of sEV by Differential Ultracentrifugation (DUC)
4.3. Isolation of sEV by Exo-spin™ Mini-HD Column EX05
4.4. Nanoparticle Tracking Analysis
4.5. Primary and Secondary Antibodies
4.6. Transmission Electron Microscopy and Immuno-Electron Microscopy
4.7. SDS-PAGE and Western Blotting
4.8. ELISA
4.9. MACPlex Human Exosome Pan Kit
4.10. Micro BCA™ Protein Assay Kit
4.11. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
Abbreviations
APC | Allophycocyanin |
BCA | Bicinchoninic acid |
BSA | Bovine serum albumin |
DF | Dilution Factor |
DUC | Differential ultracentrifugation |
EDTA | Ethylenediaminetetraacetic acid |
ELISA | Enzyme-linked immunoassay |
EV | Extracellular vesicles |
HDL | High-density lipoproteins |
HRP | Horseradish peroxidase |
LDL | Low-density lipoproteins |
MHC | Major histocompatibility complex |
NTA | Nanoparticle tracking analysis |
OPD | O-Phenylenediamine dihydrochloride |
RT | Room temperature |
sEV | Small extracellular vesicles |
TBS | Tris-buffered saline |
TBST | Tris-buffered saline + Tween20 |
TEM | Transmission electron microscopy |
TRIS | Trisaminomethane |
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Marker | DUC | Exo-spin™ |
---|---|---|
CD62P | 41,826 | 5839 |
CD41b | 38,839 | 8594 |
CD42a | 38,260 | 4270 |
CD9 | 29,016 | 5195 |
CD63 | 28,660 | 11,308 |
CD29 | 26,669 | 3089 |
HLA-ABC | 18,512 | 1518 |
HLA-DRDPDQ | 12,069 | 2206 |
CD24 | 10,192 | 957 |
CD81 | 5905 | 3764 |
CD31 | 4360 | 606 |
CD56 | 2969 | 153 |
CD105 | 2781 | 512 |
CD40 | 2759 | 451 |
CD3 | 2291 | 33 |
CD45 | 2090 | 191 |
CD69 | 1721 | 482 |
CD44 | 1522 | 525 |
CD146 | 1234 | 502 |
CD25 | 1121 | 221 |
CD142 | 1011 | 417 |
CD86 | 788 | 481 |
CD8 | 765 | 1052 |
CD14 | 679 | 75 |
CD49e | 466 | 155 |
CD326 | 369 | 360 |
ROR1 | 291 | 556 |
mIgG1 ctrl | 210 | 281 |
CD133/1 | 156 | 378 |
CD4 | 139 | 320 |
CD20 | 132 | 323 |
REA ctrl | 128 | 504 |
CD2 | 85 | 265 |
MCSP | 55 | 258 |
CD209 | 36 | 451 |
CD11c | 0 | 233 |
CD19 | −16 | 352 |
SSEA-4 | −22 | 4 |
CD1c | −198 | 161 |
Characteristics | DUC | Exo-spin™ | Comments |
---|---|---|---|
Minimal serum volume for healthy individuals | 1 mL | 100 µL | DUC range: 1–5 mL |
Exo-spin™ range: x–150 µL | |||
Final volume of sEV resuspended in PBS | 200 µL | 400 µL | Volume of DUC can be adjusted for individual purposes |
Total protein concentration | 32.4 ± 7.8 µg/mL | 40.6 ± 16.8 µg/mL | |
Recovery (particle number/mL of serum) | 1.0 ± 1.1 × 108/mL | 497.6 ± 769.5 × 108/mL | High recovery of particles by Exo-spin is mostly associated with large numbers of lipoproteins being co-isolated; |
Normalisation is essential since the initial applied volume of serum differs | |||
Absolute number of particles | 2.7 ± 2.2 × 108 | 49.8 ± 76.9 × 108 | Absolute number of particles that is purified from heathy individuals according to the suited protocol |
Particle size (diameter) ø | Mean = 148.1 ± 14.6 nm | Mean = 132.5 ± 12.1 nm | Large size of particles isolated by DUC may be associated with aggreagates induction by gravitational forces |
Mode = 121.9 ± 20.6 nm | Mode = 110.9 ± 17.2 nm | ||
Median = 138.0 ± 13.7 nm | Median = 123.1 ± 13.1 nm | ||
% of particles < 150 nm = 57.66 ± 8.8% | % of particles < 150 nm = 69.6 ± 8.4% | ||
Relative mean intensity by ELISA | CD81: 0.36 ± 0.18; | CD81: 0.12 ± 0.08; | Measured particle concentrations for these samples were: DUC = 10.54 × 108/mL; Exo-spin™ = 32.25 × 108/mL |
CD63: 0.52 ± 0.25; | CD63: 0.16 ± 0.07; | ||
CD9: 0.96 ± 0.48 | CD9: 0.18 ± 0.07 | ||
Median APC fluorescence intensity by MACSPlex | Average: 7124.8 ± 12,203.1 | Average: 1462.4 ± 2451.1 | All samples were paired |
CD81: 5905.2 ± 3686.9 | CD81: 3763.8 ± 3586.7 | ||
CD63: 28,660 ± 12,303 | CD63: 11,308 ± 8743.4 | ||
CD9: 29,016 ± 16,072 | CD9: 5194.5 ± 4542.6 | ||
Morphology | Singles and aggregates of vesicles, cup-like shape, approximately half of vesicle were stained positively with exosomal markers | Whole image fully layered with vesicles, various morphologies that does not resemble exosomes, only few stained positively with exosomal markers | - |
Co-purification | Low lipoprotein contamination | High lipoprotein contamination | Most of the particles isolated by Exo-spin™ from non-fasted patients were assigned as lipoproteins; |
Albumin contamination approximately 12.85% | Non-detectable albumin contamination | Albumin contamination was evaluated by silver staining. |
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Małys, M.S.; Aigner, C.; Schulz, S.M.; Schachner, H.; Rees, A.J.; Kain, R. Isolation of Small Extracellular Vesicles from Human Sera. Int. J. Mol. Sci. 2021, 22, 4653. https://doi.org/10.3390/ijms22094653
Małys MS, Aigner C, Schulz SM, Schachner H, Rees AJ, Kain R. Isolation of Small Extracellular Vesicles from Human Sera. International Journal of Molecular Sciences. 2021; 22(9):4653. https://doi.org/10.3390/ijms22094653
Chicago/Turabian StyleMałys, Małgorzata S., Christof Aigner, Stefan M. Schulz, Helga Schachner, Andrew J. Rees, and Renate Kain. 2021. "Isolation of Small Extracellular Vesicles from Human Sera" International Journal of Molecular Sciences 22, no. 9: 4653. https://doi.org/10.3390/ijms22094653