Isolation of small extracellular vesicles from small volumes of blood plasma using size exclusion chromatography and density gradient ultracentrifugation
- School of Biological Sciences, Nanyang Technological University, Singapore
- Facility for Analysis, Characterisation, Testing and Simulation, Nanyang Technological University, Singapore
- Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore
- Department of Material Science and Engineering, Massachusetts Institute of Technology, United States
Figures
Figure 1 with 1 supplement
Size exclusion chromatography (SEC) was effective in removing plasma proteins and high-density lipoprotein (HDL) but not low-density lipoproteins.
(A) 500 μL of plasma was loaded on a SEC column and particle fractions (PFs) were collected. (B) Cryo-EM images of PFs obtained from the plasma of five healthy individuals. Vesicles could be easily identified, and the images revealed a clean background suggesting minimum protein contamination. Moreover, HDL was sparse in the cryo-EM images, indicating that they were also largely removed. Furthermore, small extracellular vesicles (sEVs), having clearly defined bilayers (red arrows), were easily distinguishable from lipoproteins (representative particles marked by yellow arrows). The ratio of counted sEVs to all vesicles is shown under each image. All scale bars represent 200 nm.
Figure 1—figure supplement 1
Size exclusion chromatography (SEC) elution profiles, EM images of the particle fraction (PF), and particle size distributions.
(A) SEC elution profiles according to particle concentration (by nanoparticle tracking analysis [NTA]) of six different plasma sources (left) and four repeats of the same plasma source (middle). Protein concentrations (by BCA, right) in correspondence to the elution profiles of the six different plasma sources shown on the left. 500 μL of plasma was loaded onto the SEC column, and 10 fractions of 500 μL each were collected and analyzed. Fractions 7–10 were pooled to constitute the SEC-PF because of their high particle concentrations and low protein concentrations. (B) Representative transmission electron microscopy (TEM) images of the PF. (C) Cryo-EM images of the SEC-PF. (D, E) Typical particle size distributions of a plasma and SEC-PF measured by NTA. (F) A histogram of particle diameters obtained from the TEM images shown in (B).
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Figure 1—figure supplement 1—source data 1
Numerical data corresponding to size exclusion chromatography (SEC) elution profiles, nanoparticle tracking analysis (NTA) size distributions, and transmission electron microscopy (TEM)-derived particle size histograms.
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig1-figsupp1-data1-v1.xlsx
Figure 2 with 1 supplement
Design of a density gradient setup in a small volume format.
(A) 500 μL phosphate-buffered saline (PBS) was overlaid on 800 μL 10% iodixanol solution and 20 μL 50% iodixanol cushion. The 1.5 mL tubes were subjected to ultracentrifugation using a fixed-angle rotor at an average speed of 135,000×g for 2, 6, and 16 hr at 4°C to establish a density gradient profile. (B) Density gradient profiles along the 1.5 mL tubes after ultracentrifugation. The 2 hr spinning time gave a density profile with the largest separation zone of 1.05–1.08 g/mL and smallest small extracellular vesicle (sEV) zone of >1.08 g/mL. The data shows the average ± standard deviation of seven repeats for 2 hr, three repeats each for 6 and 16 hr.
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Figure 2—source data 1
Numerical data corresponding to density gradient profiles across fractions under different centrifugation durations.
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Distribution of small extracellular vesicles (sEVs) and lipoproteins within the density gradient after density gradient ultracentrifugation (DGUC).
(A) Size exclusion chromatography (SEC)-particle fractions (PFs) (6 mL) were placed on top of a density gradient cushion constructed with 2 mL 10%, 2 mL 30%, and 2 mL 50% iodixanol solutions and centrifuged at 150,000×g for 2 hr at 4°C. (B) After DGUC, the tube was fractionated into 42 fractions, which were each examined for their densities, particle concentrations (by nanoparticle tracking analysis [NTA]), and presence of sEVs and lipoproteins (by transmission electron microscopy [TEM]). The dominance of lipoproteins was evident in fractions of density <1.05 g/mL, which is designated as the LP zone. sEVs started to appear when density exceeded 1.05 g/mL but a significant level of lipoproteins was present until the density of 1.08 g/mL. Therefore, the density region of 1.05–1.08 g/mL was designated as the LP+sEV zone. Beyond 1.08 g/mL, lipoproteins diminished drastically, and the density region of >1.08 g/mL was designated as the sEV zone. All scale bars represent 200 nm.
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Figure 2—figure supplement 1—source data 1
Numerical data corresponding to density values and particle concentration across gradient fractions.
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig2-figsupp1-data1-v1.xlsx
Figure 3
Particle and protein concentrations along the 1.5 mL tube after applying density gradient ultracentrifugation (DGUC) to plasma.
(A) The 1.5 mL tube was fractionated into 13 fractions. (B) Particle concentration ± SD measured by nanoparticle tracking analysis (NTA) of different fractions (n=3). (C) Protein concentrations ± SD measured by BCA of different fractions (n=3).
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Figure 3—source data 1
Numerical data corresponding to particle concentration (NTA) and protein concentration (BCA) across fractions.
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig3-data1-v1.xlsx
Figure 4
Schematic representation of the size exclusion chromatography (SEC)-density gradient ultracentrifugation (DGUC) and DGUC-SEC protocols.
(A) Illustrates the SEC-DGUC protocol sequence. Starting from the left, SEC is used initially to separate plasma proteins and high-density lipoprotein (HDL). Subsequently, DGUC is employed to separate low-density lipoproteins (i.e. IDL, VLDL, LDL) from small extracellular vesicles (sEVs). (B) Depicts the DGUC-SEC protocol, wherein DGUC is initially employed to segregate high-density components from those of lower density, before SEC is applied to partition any remaining proteins from the sEVs.
Figure 5 with 2 supplements
Density gradient ultracentrifugation (DGUC) in the 1.5 mL tube format effectively separated small extracellular vesicles (sEVs) from lipoproteins.
Particle fractions (PFs) obtained from size exclusion chromatography (SEC) were concentrated into 500 μL, loaded onto the density gradient, and subjected to ultracentrifugation as previously described. After DGUC, the 1.5 mL tube was fractionated into 13 fractions, which were each examined for their particle concentration (by nanoparticle tracking analysis [NTA]) and the presence of sEVs and lipoproteins (by transmission electron microscopy [TEM]). In the sEV zone (bottom of the tube), where density was higher than 1.08 g/mL, high-purity sEVs were indeed observed. A mixed population of sEVs and lipoproteins was observed within the density zone of 1.05–1.08 g/mL. Interestingly, the particle numbers in this density zone were low, thus creating an effective separation zone between lipoproteins and sEVs. The RNA profiles of corresponding particle populations are shown on the right. All scale bars represent 200 nm.
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Figure 5—source data 1
Numerical data corresponding to particle concentration profiles, particle size distributions, and RNA electropherogram profiles.
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Transmission electron microscopy (TEM) images and size distributions of the 13 fractions collected from the 1.5 mL tube following size exclusion chromatography (SEC)-density gradient ultracentrifugation (DGUC).
(A) Representative TEM images of each of the 13 fractions. SEC-DGUC-1 clearly showed minimum presence of lipoprotein (high contrast lighter color particles) compared to other fractions. Moreover, the presence of small extracellular vesicles (sEVs) (low contrast and cup-shaped particles) is evident in SEC-DGUC-1. A typical image of the 1.5 mL tube after the DGUC step is shown at the left (bottom picture). The whitish layer at the top, gradually becoming clearer toward the bottom of the tube, corresponds well with the TEM observations. (B) Particle size distributions measured according to the TEM images of the 13 fractions.
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Figure 5—figure supplement 1—source data 1
Numerical data corresponding to transmission electron microscopy (TEM)-derived particle size distributions for 13 fractions.
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig5-figsupp1-data1-v1.xlsx
Figure 5—figure supplement 2
SEC-DGUC fractionation reveals efficient sEV isolation after 2h ultracentrifugation.
(A) The 1.5 mL tube was fractionated into 13 fractions. (B) Size distributions (by nanoparticle tracking analysis [NTA]) of the 13 fractions collected from the 1.5 mL tube following size exclusion chromatography (SEC)-density gradient ultracentrifugation (DGUC) protocol. The particle size distribution in SEC-DGUC-1 was distinct from the rest of the fractions. Even in SEC-DGUC-2 (highlighted in purple), the size distribution follows fractions 3–13, implying the dominance of lipoproteins in these fractions. (C) The 1.5 mL tube was fractionated into four fractions in order to examine if 2 hr spinning time was sufficient to isolate small extracellular vesicle (sEV) from lipoproteins in the 1.5 mL tube format DGUC. (D) The resulting size distributions of SEC-DGUC-1s (by NTA) were highly similar for 2 hr and 16 hr spinning time. (E) The particle concentrations of the four individual fractions along the 1.5 mL tube closely resembled each other for 2 hr and 16 hr, implying that there was no significant loss of sEV for 2 hr spinning time in comparison to 16 hr.
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Figure 5—figure supplement 2—source data 1
Numerical data corresponding to particle size distributions and particle concentration profiles under different centrifugation durations.
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig5-figsupp2-data1-v1.xlsx
Figure 6 with 1 supplement
The high purity of small extracellular vesicles (sEVs) in SEC-DGUC-1 was demonstrated by WB and cryo-EM.
(A) The 1.5 mL tube subjected to the size exclusion chromatography (SEC)-density gradient ultracentrifugation (DGUC) protocol was fractionated into four fractions for easier analysis. (B) Western blot analyses of original plasma, SEC-PF, SEC-DGUC-1–4 using sEV and lipoprotein markers. (C, D) Cryo-EM images showing the comparison between SEC-PF and SEC-DGUC-1. The SEC-DGUC-1 in (D) was obtained from non-fasting plasma collected in ethylenediaminetetraacetic acid (EDTA) tubes. All scale bars represent 200 nm.
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Figure 6—source data 1
Original uncropped western blot images for Figure 6.
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig6-data1-v1.zip
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Figure 6—source data 2
Annotated uncropped western blot images for Figure 6, indicating lane identities and bands used in the analysis.
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig6-data2-v1.zip
Figure 6—figure supplement 1
Additional cryo-EM images of SEC-DGUC-1.
Red arrows represent small extracellular vesicles (sEVs), and yellow arrows represent typical lipoproteins. The SEC-DGUC-1 was obtained from non-fasting plasma collected in ethylenediaminetetraacetic acid (EDTA) tubes. All scale bars represent 200 nm.
Figure 7
Comparison of small extracellular vesicles (sEVs) isolated by size exclusion chromatography (SEC)-density gradient ultracentrifugation (DGUC) and DGUC-SEC protocols.
(A) Western blot of SEC-DGUC-1, DGUC-SEC-PF, sEV isolated by differential ultracentrifugation (dUC) obtained from 2 mL of plasma of the same source. (B) Particle size distributions in SEC-DGUC-1 and DGUC-SEC-PF measured by transmission electron microscopy (TEM). (C, D) TEM images of SEC-DGUC-1 and DGUC-SEC-PF. (E, F) Total RNA analyses of SEC-DGUC-1 and DGUC-SEC-PF. Note that data in (E) is the same data shown in the bottom right subfigure of Figure 5.
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Figure 7—source data 1
Numerical data corresponding to particle diameter distributions derived from transmission electron microscopy (TEM) images and RNA electropherogram profiles.
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig7-data1-v1.xlsx
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Figure 7—source data 2
Original uncropped western blot images for Figure 7.
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig7-data2-v1.zip
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Figure 7—source data 3
Annotated uncropped western blot images for Figure 7, indicating lane identities and bands used in the analysis.
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig7-data3-v1.zip
Figure 8 with 1 supplement
Flow cytometry assay using MACSPlex Exosome Kit to evaluate small extracellular vesicle (sEV) isolates.
(A) Illustration of the principle of MACSPlex Exosome Kit. 37 types of beads with different fluorescent colors are functionalized with specific antibodies against sEV surface proteins. sEVs captured by the beads are detected with flow cytometry using APC-labeled anti-CD9, CD63, CD81 antibodies. (B) Flow cytometry gating setup. (C) A representative data showing the comparison of median fluorescence intensity (MFI) ± standard error of the mean (SEM) from sEV isolates obtained from SEC-PF and SEC-DGUC-1. Equal numbers of particles (5×108, based on nanoparticle tracking analysis [NTA]) were loaded for both samples. (D) Comparison of signal patterns among plasma, SEC-DGUC-1, SEC-PF, and differential ultracentrifugation (dUC). The results showed that sEVs isolated from dUC deviate from plasma, whereas sEVs in the SEC-PF and SEC-DGUC-1 mirror the sEV population in plasma.
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Figure 8—source data 1
Numerical data corresponding to flow cytometry analysis of surface marker expression.
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig8-data1-v1.xlsx
Figure 8—figure supplement 1
Flow cytometry data of size exclusion chromatography (SEC)-particle fraction (PF) vs. SEC-DGUC-1 obtained from two plasma sources using MACSPlex Exosome Kit.
Data from two plasma sources demonstrated a similar trend of much stronger signals of SEC-DGUC-1 compared to SEC-PF in flow cytometry using MACSPlex Exosome Kit. Data are median ± SEM.
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Figure 8—figure supplement 1—source data 1
Numerical data corresponding to flow cytometry analysis across plasma samples.
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig8-figsupp1-data1-v1.xlsx
Figure 9
Functional annotation of proteins identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS).
(A) Functional annotation of proteins identified in size exclusion chromatography (SEC)-DGUC-1. (B) Functional annotation of proteins identified in SEC-particle fraction (PF).
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Figure 9—source data 1
Numerical data corresponding to functional annotation and enrichment analysis of proteins identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS).
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig9-data1-v1.xlsx
Figure 10 with 2 supplements
Repeatability of the size exclusion chromatography (SEC)-density gradient ultracentrifugation (DGUC) protocol.
(A) Particle concentration profiles (by nanoparticle tracking analysis [NTA]) along the 1.5 mL tube from four technical replicates subjected to SEC-DGUC protocol. (B) Particle size distributions measured by NTA in the four fractions indicated on the left figure. Four technical replicates subjected to SEC-DGUC protocol were shown. Jensen-Shannon divergence (JSD) values for SEC-DGUC-1–4 are 0.015, 0.006, 0.001, and 0.002, indicating strong similarities among the histograms.
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Figure 10—source data 1
Numerical data corresponding to particle concentration profiles and size distributions from repeatability experiments.
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig10-data1-v1.xlsx
Figure 10—figure supplement 1
Repeatability of size exclusion chromatography (SEC).
(A–C) Particles size distributions of SEC-particle fraction (PF) measured by nanoparticle tracking analysis (NTA). (A, B, and C) correspond to the first, second, and third rows of the table, respectively. The highly overlapped size distributions allude to the consistency in the particles eluted from SEC columns, even though the number of particles eluted varied up to 55%. The table lists six experiments with various numbers of repeats of SEC. The average particle concentrations, standard deviation, and coefficient of variation (CV) (SD/average ratio) are listed together with the number of measurements made.
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Figure 10—figure supplement 1—source data 1
Numerical data corresponding to particle size distributions and summary statistics from repeatability experiments.
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig10-figsupp1-data1-v1.xlsx
Figure 10—figure supplement 2
Repeatability of size exclusion chromatography (SEC)-density gradient ultracentrifugation (DGUC) protocol.
Nanoparticle tracking analysis (NTA) measurements of two experiments to test repeatability after SEC-DGUC. The figure displays the size distributions measured by NTA of the experiment listed in the second row of the table. The size distributions of the experiment listed in the first row of the table were presented in main Figure 10. The table lists the average particle concentrations of SEC-DGUC-1 and the standard deviation, coefficient of variation (CV) (SD/average ratio) together with the number of measurements made.
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Figure 10—figure supplement 2—source data 1
Numerical data corresponding to particle size distributions from repeatability experiments.
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig10-figsupp2-data1-v1.xlsx
Figure 11 with 2 supplements
Reliability of the size exclusion chromatography (SEC)-density gradient ultracentrifugation (DGUC) protocol.
Transmission electron microscopy (TEM) images of SEC-DGUC-1 were obtained from five fasting plasma (biobank samples, ethylenediaminetetraacetic acid [EDTA] tubes). High purity of small extracellular vesicles (sEVs) represented by the low-contrast, cup-shaped vesicles was evident across different samples, suggesting the SEC-DGUC was reliable in obtaining high-purity sEVs. All scale bars represent 200 nm.
Figure 11—figure supplement 1
Reliability of particle concentration profiles along the 1.5 mL tube (by nanoparticle tracking analysis [NTA]).
(A) Detailed particle concentration profiles (13 fractions) of four different samples along with the calibrated density profile shown in Figure 2. (B) Five particle concentration profiles (four fractions shown in Figure 10B) in size exclusion chromatography (SEC)-DGUC-1 corresponding to the biobank plasma samples (ethylenediaminetetraacetic acid [EDTA] tubes) shown in Figure 11. Note that the data from (A) and (B) are from different plasma sources.
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Figure 11—figure supplement 1—source data 1
Numerical data corresponding to particle concentration profiles across fractions for multiple plasma samples.
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig11-figsupp1-data1-v1.xlsx
Figure 11—figure supplement 2
Size distributions (by nanoparticle tracking analysis [NTA]) of size exclusion chromatography (SEC)-DGUC-1 obtained from five fasting plasma corresponding to Figure 11.
The particle size distributions did not overlap, reflecting the variation of small extracellular vesicles (sEV) population among different plasma sources.
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Figure 11—figure supplement 2—source data 1
Numerical data corresponding to particle size distributions of size exclusion chromatography (SEC)-DGUC-1 across plasma samples.
- https://cdn.elifesciences.org/articles/92796/elife-92796-fig11-figsupp2-data1-v1.xlsx
Tables
Table 1
Particle numbers in different steps during the size exclusion chromatography (SEC)-density gradient ultracentrifugation (DGUC) protocol, calculated based on nanoparticle tracking analysis (NTA) measurement.
The first column provides the total particle numbers measured in 500 μL fasting plasma (~1 mL of whole blood) from five individuals. The second column provides the corresponding total vesicle numbers in SEC-particle fraction (PF) after the plasma was subjected to SEC. The third column provides the corresponding total vesicle numbers in SEC-DGUC-1 after the SEC-PF was subjected to DGUC.
| Plasma (particle number) | SEC-PF (particle number) | SEC-DGUC-1 (particle number) |
|---|---|---|
| 8×1010 | 6.5×1010 | 1.8×109 |
| 5.5×1011 | 1.65×1011 | 1.32×109 |
| 1.75×1011 | 8.5×1010 | 1.68×109 |
| 6×1011 | 1.2×1011 | 1.56×109 |
| 2.4×1010 | 1.6×1010 | 1.2×109 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Commercial assay or kit | ACD-A vacuette tube | Greiner, Austria | 455055 | |
| Commercial assay or kit | SEC columns | HansaBioMed Life Sciences, Tallinn, Estonia | HBM-PEV | 5 mL capacity |
| Chemical compound, drug | Iodixanol | Sigma-Aldrich | OptiPrep, D1556 | |
| Other | Ultracentrifuge tube | Beckman Coulter | 357448 | |
| Commercial assay or kit | BCA protein assay kit | Pierce, Thermo Fisher | 23225 | |
| Other | ZetaView | Particle Metrix | PMX 120 | |
| Antibody | Mouse monoclonal anti-CD63 | Santa Cruz Biotechnology, Dallas, TX, USA | sc-5275; clone MX-49.129.5 RRID:AB_627877 | WB (1:1000) |
| Antibody | Rabbit monoclonal anti-CD9 | Abcam, Cambridge, MA, USA | ab92726; clone 9EPR2949 RRID:AB_10561589 | WB (1:1000) |
| Antibody | Mouse monoclonal anti-CD81 | Santa Cruz Biotechnology, Dallas, TX, USA | sc-166029; clone B-11 RRID:AB_2275892 | WB (1:1000) |
| Antibody | Rabbit polyclonal anti-Flotillin-1 | Abcam, Cambridge, MA, USA | ab41927 RRID:AB_941621 | WB (1:1000) |
| Antibody | Rabbit polyclonal anti-TSG101 | Abcam, Cambridge, MA, USA | ab30871 RRID:AB_2208084 | WB (1:1000) |
| Antibody | Mouse monoclonal anti-ApoA-I | Santa Cruz Biotechnology, Dallas, TX, USA | sc-376818; clone B-10 RRID:AB_2797313 | WB (1:1000) |
| Antibody | Mouse monoclonal anti-ApoB | Santa Cruz Biotechnology, Dallas, TX, USA | sc-13538; clone C1.4 RRID:AB_626690 | WB (1:1000) |
| Antibody | Mouse monoclonal anti-Albumin | Abcam, Cambridge, MA, USA | ab10241 RRID:AB_296978 | WB (1:1000) |
| Antibody | Rabbit polyclonal anti-Calnexin | Abcam, Cambridge, MA, USA | ab22595 RRID:AB_2069006 | WB (1:1000) |
| Antibody | IRDye 800CW anti-mouse | Li-COR Biosciences, Lincoln, NE, USA | 925-32210 RRID:AB_2687825 | Secondary antibody, WB (1:15,000) |
| Antibody | IRDye 680LT anti-rabbit | Li-COR Biosciences, Lincoln, NE, USA | 926-68021 RRID:AB_10706309 | Secondary antibody, WB (1:15,000) |
| Other | Carbon-coated grid | Electron Microscopy Sciences, Hatfield, PA, USA | CF300-CU | |
| Commercial assay or kit | miRNeasy Serum/Plasma Kit | QIAGEN, GmbH, Hilden, Germany | 217184 | |
| Commercial assay or kit | MACSPlex Exosome Kit | Miltenyi Biotec, Bergish Gladbach, Germany | N/A | |
| Software, algorithm | ImageStudio v5.2 | N/A | ||
| Software, algorithm | Proteome Discoverer v2.1 | Thermo Fisher Scientific | N/A | |
| Other | Xbridge C18 column | Waters, Milford, MA, USA | 4.6 × 250 mm |
Additional files
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Supplementary file 1
LC-MS/MS proteins identified in SEC-DGUC-1 and SEC-particle fraction (PF) ranked according to emPAI values.
(a) Liquid chromatography with tandem mass spectrometry (LC-MS/MS) proteins identified in size exclusion chromatography (SEC)-DGUC-1 by LC-MS/MS analysis ranked according to emPAI values. (b) LC-MS/MS proteins identified in SEC-particle fraction (PF) by LC-MS/MS analysis ranked according to emPAI values.
- https://cdn.elifesciences.org/articles/92796/elife-92796-supp1-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/92796/elife-92796-mdarchecklist1-v1.docx
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Isolation of small extracellular vesicles from small volumes of blood plasma using size exclusion chromatography and density gradient ultracentrifugation
eLife 13:RP92796.
https://doi.org/10.7554/eLife.92796.3
