Framework for rapid comparison of extracellular vesicle isolation methods
- Wyss Institute for Biologically Inspired Engineering, United States
- Tufts University School of Medicine, United States
- Department of Pathology, Brigham and Women’s Hospital, United States
- Harvard Medical School, United States
Figures
Figure 1
Overview of experimental framework for EV detection using Simoa and size exclusion chromatography (SEC).
(a) Different methods of EV isolation can be directly compared to assess yield and purity by measuring the three tetraspanins (CD9, CD63, and CD81) and albumin. (b) Single immuno-complexes are …
Figure 2 with 1 supplement
Comparison of existing methods for EV isolation in plasma and CSF.
(a) Schematic of experimental outline. (b–d) Individual tetraspanin yields using different isolation methods from plasma. (e) Relative EV recoveries from plasma were calculated by first normalizing …
-
Figure 2—source data 1
Comparison of existing methods for EV isolation in plasma and CSF.
- https://cdn.elifesciences.org/articles/70725/elife-70725-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Assay reproducibility between Simoa measurements of EV isolations on 2 different days.
Simoa results displaying average measurements of EV isolation on two different days with error bars representing standard deviation. (a–d) for tetraspanins and albumin for EV isolation in plasma. (e–…
Figure 3 with 5 supplements
Comparison of SEC methods for EV isolation in plasma.
(a) Levels of tetraspanins and albumin in plasma after fractionation with 10 ml custom columns filled with Sepharose CL-6B (top), Sepharose CL-4B (middle), and Sepharose CL-2B (bottom). (b) Levels …
-
Figure 3—source data 1
Plasma SEC optimization.
- https://cdn.elifesciences.org/articles/70725/elife-70725-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Custom stand designed for higher throughput, reproducible SEC.
(a) Image of SolidWorks file with custom SEC stand designed to run four SEC columns in parallel with ‘clickable’ sliding collection tube holder plates that allow for easy fraction collection. (b) …
Figure 3—figure supplement 2
Comparison of SEC resins by Western blotting.
(a–c) Western blot of tetraspanins in fractions 7–10 of 0.5 ml plasma isolated by SEC using Sepharose CL-2B, Sepharose CL-4B, and Sepharose CL-6B resins. SEC, size exclusion chromatography.
Figure 3—figure supplement 3
Effect of plasma sample volume on SEC.
Simoa was performed to determine levels of CD9, CD63, CD81, and albumin after fractionating different volumes of plasma by SEC using a 10 mL Sepharose CL-6B column. Effect of sample volume on EV …
Figure 3—figure supplement 4
Comparison of EV recovery and albumin contamination across all tested methods in plasma.
Comparison of plasma EV recovery and albumin contamination in plasma across all tested methods ranked by EV recovery. Relative EV recoveries were calculated by individually normalizing each …
Figure 3—figure supplement 5
Coefficients of variation (CVs) across all tested methods in plasma.
Figure 4 with 3 supplements
Comparison of SEC methods for EV isolation in CSF.
(a) Levels of tetraspanins and albumin in CSF after fractionation with 10 ml custom columns filled with Sepharose CL-6B (top), Sepharose CL-4B (middle), and Sepharose CL-2B (bottom). (b) Levels of …
-
Figure 4—source data 1
CSF SEC optimization.
- https://cdn.elifesciences.org/articles/70725/elife-70725-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Effect of CSF sample volume on SEC.
Simoa was performed to determine levels of CD9, CD63, CD81, and albumin after fractionating different volumes of CSF by SEC using a 10-ml Sepharose CL-6B column. Effect of sample volume on EV …
Figure 4—figure supplement 2
Comparison of EV recovery and albumin contamination across all tested methods in CSF.
Comparison of plasma EV recovery and albumin contamination in CSF across all tested methods ranked by EV recovery. Relative EV recoveries were calculated by individually normalizing each tetraspanin …
Figure 4—figure supplement 3
Coefficients of variation (CVs) across all tested methods in CSF.
Figure 5
Comparison of top custom SEC methods in plasma and CSF.
Error bars represent the standard deviations from four replicates of each column. (a–c). Individual tetraspanin yields using different isolation methods from plasma. (d) Relative EV recoveries from …
-
Figure 5—source data 1
Top SEC methods in new batches of plasma and CSF.
- https://cdn.elifesciences.org/articles/70725/elife-70725-fig5-data1-v2.xlsx
Tables
Table 1
Recommendations for SEC columns for EV isolation from plasma and CSF.
| High yield | High purity | |
|---|---|---|
| Plasma | Sepharose CL-6B10 ml column fractions 7–10 | Sepharose CL-4B20 ml column fractions 14–17 |
| CSF | Sepharose CL-6B10 ml column fractions 7–10 | Sepharose CL-4B10 ml column fractions 7–10 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Biological sample (human) | Plasma | BioIVT | Cat #HUMANPLK2PNN | Pooled gender, K2EDTA |
| Biological sample (human) | Cerebrospinal fluid | BioIVT | Cat# HMNCSFR-NODXR | Pooled gender, no diagnosis remnant |
| Antibody | Anti-CD9 (Mouse monoclonal) | MilliporeSigma | Cat# CBL162RRID:AB_2075914 | WB (1:1000) |
| Antibody | Anti-CD9 (Rabbit monoclonal) | Abcam | Cat# ab195422RRID:AB_2893477 | Simoa capture |
| Antibody | Anti-CD9 (Mouse monoclonal) | Abcam | Cat# ab58989RRID:AB_940926 | Simoa detector |
| Antibody | Anti-CD63 (Mouse monoclonal) | BD | Cat# 556019RRID:AB_396297 | Simoa detector;WB (1:1000) |
| Antibody | Anti-CD63 (Mouse monoclonal) | R&D Systems | Cat# MAB5048RRID:AB_2275726 | Simoa capture |
| Antibody | Anti-CD81 (Mouse monoclonal) | Thermo Fisher Scientific | Cat# 10630DRRID:AB_2532984 | WB (1:666) |
| Antibody | Anti-CD81 (Mouse monoclonal) | Abcam | Cat# ab79559RRID:AB_1603682 | Simoa capture |
| Antibody | Anti-CD81 (Mouse monoclonal) | BioLegend | Cat# 349502RRID:AB_10643417 | Simoa detector |
| Commercial assay or kit | Human Serum Albumin DuoSet ELISA | R&D Systems | Cat# DY1455 | Simoa capture and detector |
| Peptide, recombinant protein | CD9 | Abcam | Cat# ab152262 | |
| Peptide, recombinant protein | CD63 | Origene | Cat# TP301733 | |
| Peptide, recombinant protein | CD81 | Origene | Cat# TP317508 | |
| Peptide, recombinant protein | Albumin | Abcam | Cat# ab201876 | |
| Commercial assay or kit | ExoQuick exosome precipitation solution | SBI | Cat# EXOQ5A-1 | |
| Commercial assay or kit | ExoQuick ULTRA EV isolation kit for plasma and serum | SBI | Cat# EQULTRA-20A-1 | |
| Commercial assay or kit | qEVoriginal 70 nm | Izon | Cat# SP1 | |
| Commercial assay or kit | qEVoriginal 35 nm | Izon | Cat# SP5 | |
| Other | Sepharose CL-2B | Cytiva | Cat# 17014001 | |
| Other | Sepharose CL-4B | Cytiva | Cat# 17015001 | |
| Other | Sepharose CL-6B | Cytiva | Cat# 17016001 |
Additional files
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/70725/elife-70725-transrepform1-v2.docx
-
Source data 1
All data combined.
- https://cdn.elifesciences.org/articles/70725/elife-70725-supp1-v2.xlsx
