Viscosity of Plasma as a Key Factor in Assessment of Extracellular Vesicles by Light Scattering
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.1.1. Exosome Standards
2.1.2. Blood Plasma Samples
2.1.3. Blood Plasma for the Ultracentrifugation Experiments and Viscosity Determination
2.1.4. Human Serum Albumin
2.2. Methods
2.2.1. Static and Dynamic Light Scattering Measurements
2.2.2. Viscosity and Density Measurements
2.2.3. Asymmetric Flow Field Flow Fractionation (AF4/UV-MALS)
2.2.4. Flow Cytometry (FCM)
2.2.5. Atomic Force Microscopy (AFM)
3. Results
3.1. LS Size Characterization of the Exosome Standard
3.2. LS Size Characterization of Particles in Blood Plasma
3.3. Determination of the Medium Viscosity for Correct Characterization of the EV size by DLS in Full Blood Plasma Samples
3.4. Shape Characterization of Particles by DLS/SLS
4. Discussion
4.1. Size Characterization
4.2. Identification of Subpopulations of Extracellular Vesicles in Blood Plasma
4.3. Structural Characterization
4.4. Further Considerations
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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AF4-MALS | Batch DLS/SLS | |||
---|---|---|---|---|
Sample | Rg (nm) | Rh,90 # (nm) | Rg (nm) | |
unfiltered ES | larger EVs (population 2) | 93 | 119 | 152 |
smaller EVs (population 1) | ~40 | 20 | ND * | |
Average of short-time eluted particles; i.e., components with high UV signal and smaller EVs (population 1) | ~25 | |||
filtered ES | larger EVs (population 2) | 84 | 101 | 138 |
smaller EVs (population 1) | ~40 | 19 | ND * | |
Average of short-time eluted particles; i.e., components with high UV signal and smaller EVs (population 1) | ~25 |
Peak | I [%] | Rh,90 [nm] | Rh,90corr [nm] | Measured Viscosity, η [mPa/s] | |
---|---|---|---|---|---|
Initial plasma sample I (total) = 792 kHz/mW | 0 | 5 (a) | 3 (a) | 3 (a) | 1.47 |
1 | 31 | 19 | 12 | ||
2 | 64 | 75 | 46 | ||
Colourless supernatant I (total) = 904 kHz/mW | 1 | 46 | 21 | 20 | 0.94 |
2 | 54 (b) | 55 (b) | 52 (b) | ||
Yellow supernatant I (total) = 746 kHz/mW | 0 | 3 (a) | 2 (a) | 2 (a) | 1.32 |
1 | 31 | 14 | 9 | ||
2 | 64 | 62 | 42 | ||
Whitish pellet re-suspended in PBS I (total) = 490 kHz/mW | 1 | 17 (a) | 7 (a) | 7 (a) | NC c) |
2 | 77 | 44 | 44 | ||
Gelatinous pellet re-suspended in PBS I (total) = 113 kHz/mW | 0 | 50 (a) | 7 (a) | 7 (a) | NC (c) |
1 | 49 | 40 | 40 | ||
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Božič, D.; Sitar, S.; Junkar, I.; Štukelj, R.; Pajnič, M.; Žagar, E.; Kralj-Iglič, V.; Kogej, K. Viscosity of Plasma as a Key Factor in Assessment of Extracellular Vesicles by Light Scattering. Cells 2019, 8, 1046. https://doi.org/10.3390/cells8091046
Božič D, Sitar S, Junkar I, Štukelj R, Pajnič M, Žagar E, Kralj-Iglič V, Kogej K. Viscosity of Plasma as a Key Factor in Assessment of Extracellular Vesicles by Light Scattering. Cells. 2019; 8(9):1046. https://doi.org/10.3390/cells8091046
Chicago/Turabian StyleBožič, Darja, Simona Sitar, Ita Junkar, Roman Štukelj, Manca Pajnič, Ema Žagar, Veronika Kralj-Iglič, and Ksenija Kogej. 2019. "Viscosity of Plasma as a Key Factor in Assessment of Extracellular Vesicles by Light Scattering" Cells 8, no. 9: 1046. https://doi.org/10.3390/cells8091046