The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.
Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.
Extracellular vesicles (EVs) have been proposed to define cell-released lipid bilayer-enclosed extracellular structures1, which play important roles in various physiological and pathological events2. EVs released by healthy cells can be broadly divided into two main categories, namely exosomes (or small EVs) formed through an intracellular endocytic trafficking pathway3 and microvesicles (or large EVs) developed by the outward budding of the plasma membrane of the cell4. While many studies focus on the function of EVs collected from cultured cells in vitro5, EVs derived from the circulation or tissues are more complex and heterogeneous, which have the advantage of reflecting the true state of the organism in vivo6. Furthermore, nearly all kinds of tissues can produce EVs in vivo, and these EVs can act as messengers within the tissue or be transferred by various body fluids, especially the peripheral blood, to facilitate systemic communication7. EVs in the circulation and tissues are also targets for disease diagnosis and treatment8.
Whereas exosomes have been intensively studied in recent years, microvesicles also have important biological functions, which can be easily extracted without ultracentrifugation, thus promoting basic and clinical research9. Notably, a critical issue regarding EVs isolated from the circulation and tissues is that they are derived from different cell types10. Since microvesicles are blebbed from the plasma membrane and featured by an abundance of cell surface molecules9, using parent cell membrane markers to identify the cellular origin of these EVs is feasible. Specifically, the flow cytometry (FC) technique can be applied to detect membrane markers. Moreover, researchers can isolate the EVs and make further analyses based on the functional cargos.
The present protocol provides a thorough procedure for extracting and characterizing EVs from in vivo samples. The EVs are isolated via differential centrifugation, and the characterization of EVs includes morphological identification via nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), origin analysis via FC, and protein cargo analysis via western blot. The blood plasma and maxillary bone of mice are used as representatives. Researchers can refer to this protocol for EVs from other sources and make corresponding modifications.
The animal experiments were performed in accordance with the Guidelines of Institutional Animal Care and Use Committee of the Fourth Military Medical University and the ARRIVE guidelines. For the present study, 8-week-old C57Bl/6 mice (no preference for either females or males) were used. The steps involved in isolating plasma and tissue EVs are illustrated in Figure 1. The plasma is taken as a representative to describe the EV isolation procedure from body fluids. The maxillary bone is taken as a representative to explain the EV isolation procedure from the solid tissues.
1. Preparation of the plasma and the maxillary bone samples
2. Extraction of EVs
3. Morphological identification of EVs
4. Origin analysis of EVs
NOTE: The identification of the cellular origin of EVs requires the application of antibodies for typical cell membrane markers. The minimum plasma volume to extract the EVs for the below procedure is 300 µL. Based on the lipid bilayer structures of EVs, membrane dye can be used to mark them. For blood plasma samples, CD18 for lymphocytes14 is chosen for representation. For maxillary bone samples, osteoclast-associated receptor (OSCAR) for osteoclasts15 is selected as an example. Before the FC, ensure the flow cytometer is adapted to the measurement of EVs16, as the lower size limit is largely different between distinct flow cytometers.
5. Protein content analysis of EVs
NOTE: The analysis of the protein content within EVs is performed via western blot. For example, the plasma and bone EVs were selected to analyze 6-phosphogluconate dehydrogenase (PGD) and pyruvate kinase M2 (PKM2) for metabolic status. Golgin84 (the Golgi organelle) was used as a negative control, and Flotillin (membrane protein), Caveolin (integral protein of caveolae), and β-actin (the cytoskeleton)17 were used as a positive control in EVs compared to cell samples. Mitofilin and α-Actinin-4 were chosen as large EV markers, while CD9 and CD81 were chosen as small EV markers to demonstrate the EV subpopulations18. Apoa1 was selected as a plasma lipoparticle marker19. For the respective reagent details, see Table of Materials.
According to the experimental workflow, EVs can be extracted from the peripheral blood and solid tissues (Figure 1). The maxillary bone of a mouse aged 8 weeks is approximately 0.1 ± 0.05 g, and about 300 µL of plasma can be collected from the mouse. Following the protocol steps, 0.3 mg and 3 µg of EVs can be collected, respectively. As analyzed by TEM and NTA, the typical morphological characteristics of EVs are round cup-shaped membrane vesicles with a diameter ranging from 50-300 nm (Figure 2). FC can detect the membrane dye-labeled EVs under useful controls, and FC analysis shows the percentages of specific membrane markers expressed on EVs that implies their origin from parent cells (Figure 3). Western blot analysis shows the expression of PGD and PKM2, respectively, as a representative of protein content in plasma EVs and bone EVs, indicating the metabolic status (Figure 4). Negative expression of Golgin84 in EVs is also detected with the presence of Mitofilin, α-Actinin-4, Flotillin-1, Caveolin-1, and β-actin, which is commonly enriched in plasma membrane-derived large EVs (microvesicles). The small EV marker CD9 can also be detected, while CD81 expression is low or absent, with a lack of Apoa1 existence in the plasma EVs (Figure 4).
Figure 1: Illustration of the protocol to extract EVs. (A) The steps to extract peripheral blood plasma EVs with differential centrifugation. (B) The steps to extract tissue EVs with differential centrifugation. Please click here to view a larger version of this figure.
Figure 2: Morphological identification of EVs. (A) Representative transmission electron microscope (TEM) image displaying the morphology of EVs. Scale bar = 200 nm. (B) Representative nanoparticle tracking analysis (NTA) images and quantification results show the size distribution and particle number of EVs. Please click here to view a larger version of this figure.
Figure 3: Origin analysis of EVs. (A) The different sets of tubes with different controls of the experiment. (B) The distribution of 1 µm, 0.5 µm, and 0.2 µm size beads showed in the scatted graph. (C) Representative density graphs showing PBS (tube A), the simple staining of EVs (tube B), the simple staining of membrane dye (tube C), and double staining of the EV samples (tube D). (D) Flow cytometric analysis of the percentage of CD18 positive EVs in blood plasma EVs in red compared with the secondary antibody only control in black. (E) Flow cytometric analysis of the percentage of OSCAR positive EVs from bone samples in red compared with the secondary antibody only control in black. Please click here to view a larger version of this figure.
Figure 4: Protein content analysis of EVs. (A) Representative western blot of plasma EV images showing the presence of 6-phosphogluconate dehydrogenase (PGD) and Mitofilin, α-Actinin-4, CD9, Flotillin-1, Caveolin-1, and β-actin in plasma EVs and the negative expression of CD81, Golgin84, and Apoa1. (B) Representative western blot of bone EV images showing the presence of pyruvate kinase M2 (PKM2) and Mitofilin, α-Actinin-4, CD9, CD81, Flotillin-1, Caveolin-1, and β-actin in bone EVs and the negative expression of Golgin84. Please click here to view a larger version of this figure.
Number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
BSA (µL) | 0 | 1 | 2 | 4 | 8 | 12 | 16 | 20 |
NS (µL) | 20 | 19 | 18 | 16 | 12 | 8 | 4 | 0 |
Table 1: Standard working solution of BCA assay. Add the 0.5 mg/mL of BSA and 0.9% of NS into three duplicated wells, as shown in the table.
When studying the features, the fate, and the function of EVs, it is crucial to isolate EVs with high yield and low contamination. Various methods exist to extract EVs, such as density gradient centrifugation (DGC), size-exclusion chromatography (SEC), and immunocapture assays4,20. Here, one of the most commonly used methods, differential centrifugation, was used; the advantages of this are that it is not time consuming, it generates a high yield of EVs with easy isolation from limited samples, and the ability to modify the method based on the sample source used. For analyzing the function of circulating EVs, it’s better to choose the blood plasma, which can better reflect the real state of EVs in vivo, rather than the serum. This is because many platelet-derived EVs will be released during the process of clot formation for collecting serum21, while platelets are removed via centrifugation before isolation of EVs from the plasma22. It must be noted that the choice of anticoagulation for plasma EV isolation is still controversial. The anticoagulant mechanism of heparin is more closely related to physiology; the EVs isolated thus might be more reflective of the in vivo status. It must be mentioned that heparin will cause false negative PCR reads and block EV uptake by cells23,24. Other anticoagulation reagents, such as EDTA or sodium citrate, also have their shortages, including biotoxicity, affecting the chemical properties of plasma and the platelets. Collectively, given the above information, heparin is selected in this study. To remove the platelets, the samples were centrifuged at the speed of 2,500 x g before 16,800 x g, which may remove some large EVs. Additionally, considering that many EVs within the viscous plasma will attach to platelets and get removed, it is suggested to dilute the plasma with an equal volume of PBS before removing platelets through centrifugation to improve the yield of EVs. Of note, it’s better to use a sucrose gradient with ultracentrifugation to purify the EVs to remove the soluble protein contaminations, such as protein polymers21.
To date, the common methods of characterizing EVs include NTA, TEM, FC16, and western blot, among others25. In addition, because of the alteration of morphology and quantity of EVs after being restored in different conditions13, we suggest processing the detection as soon as possible after extraction of the EVs. Besides, Görgens et al. have recommended to use PBS supplemented with human albumin and trehalose (PBS-HAT) for better preservation than pure PBS26. Morphological identification of EVs via NTA and TEM must be performed immediately after the extraction procedure, otherwise the shape of EVs may change27. Furthermore, Cryo-EM can be used28 for advanced morphological observation of EVs, which has the benefits of better preserving the shape with higher resolution. Besides NTA, the concentration of particles can also be quantified by FC with the help of a known amount of counting beads29, but this method is not very stable, and the sensitivity to small particles is low for FC. Moreover, because of the swarm detection of small particles with FC30, it’s better to use NTA for size determinations. Similarly, the origin of EVs can also be analyzed by NTA with fluorescence filters. Compared with FC, NTA is more sensitive to smaller particles and has the potential advantage of recycling the sample for other experiments. One advantage of using FC is that the specific subtypes of EVs can be enriched with fluorochrome-conjugated membrane antibodies28. Also, the function of specific surface antigens can be studied via loss-of-function experiments, such as using neutralizing antibodies28. Alternatively, 1% Triton X-100 can be used to destroy the lipid raft of EVs membrane, thus as a control for detecting membrane signals31. Furthermore, researchers can use the specific membrane characteristics to create targeted drug carriers32.
Western blot is extensively used for analyzing the protein contents in EVs. Several markers such as Mitofilin, caveolin, and α-Actinin-418 have been recommended as candidates to identify large EVs in recent studies; CD9 is enriched in small EVs rather than in large EVs, and CD81, CD63, and Syntenin-1 are extensively abundant in small EVs18. As for the results of the Western blot, markers such as Mitofilin and CD9 are variable in different animal conditions. It can be assumed that the isolated EVs represent a mixed population, in which large EVs are one major subpopulation. To verify the purity of EVs, it is suggested that the detection of organelle/nuclear proteins, such as Lamin B or Golgin84, be added as the negative control. As for plasma EVs, according to our results, Apoa1 representing lipoparticles is not enriched in the collected EVs. One limitation of this method is that it is hard to distinguish the proteins on the surface from those within the vesicle. Therefore, enzyme-linked immunosorbent assay (ELISA) can be applied for membrane protein analysis as supplementary experiments. Moreover, with the development of high-throughput sequencing, researchers can take advantage of proteomic mapping33 to analyze the protein composition of EVs, with the target proteins also needing to be verified by western blot.
In summary, this study provides a feasible protocol to isolate and characterize circulatory and tissue EVs, including an analysis of their cell sources and protein contents, which helps to establish a basis for further functional experiments.
The authors have nothing to disclose.
This work was supported by grants from the National Natural Science Foundation of China (32000974, 81870796, 82170988, and 81930025) and the China Postdoctoral Science Foundation (2019M663986 and BX20190380). We are grateful for the assistance of the National Experimental Teaching Demonstration Center for Basic Medicine (AMFU).
4% paraformaldehyde | Biosharp | 143174 | Transmission electron microscope |
Alexa fluor 488 anti-goat secondary antibody | Yeason | 34306ES60 | Flow cytometry |
Alexa fluor 488 anti-rabbit secondary antibody | Invitrogen | A11008 | Flow cytometry |
Anti-CD18 antibody | Abcam | ab131044 | Flow cytometry |
Anti-CD81 antibody | Abcam | ab109201 | Western blot |
anti-CD9 antibody | Huabio | ET1601-9 | Western blot |
Anti-Mitofilin antibody | Abcam | ab110329 | Western blot |
APOA1 Rabbit pAb | Abclone | A14211 | Western blot |
BCA protein assay kit | TIANGEN | PA115 | Western blot |
BLUeye Prestained Protein Ladder | Sigma-Aldrich | 94964-500UL | Western blot |
Bovine serum albumin | MP Biomedical | 218072801 | Western blot |
Caveolin-1 antibody | Santa Cruz Biotechnology | sc-53564 | Western blot |
CellMask Orange plasma membrane stain | Invitrogen | C10045 | Flow cytometry |
Chemiluminescence | Amersham Biosciences | N/A | Western blot |
Curved operating scissor | JZ Surgical Instrument | J21040 | EV isolation |
Electronic balance | Zhi Ke | ZK-DST | EV isolation |
Epoch spectrophotometer | BioTek | N/A | Western blot |
Eppendorf tubes | Eppendorf | 3810X | EV isolation |
Flotillin-1 antibody | PTM BIO | PTM-5369 | Western blot |
Gel imaging system | Tanon | 4600 | Western blot |
Golgin84 | Novus | nbp1-83352 | Western blot |
Grids – Formvar/Carbon Coated – Copper 200 mesh | Polysciences | 24915 | Transmission electron microscope |
Heparin Solution | StemCell | 7980 | EV isolation |
Liberase Research Grade | Sigma-Aldrich | 5401127001 | EV isolation |
Microscopic tweezer | JZ Surgical Instrument | JD1020 | EV isolation |
NovoCyte flow cytometer | ACEA | N/A | Flow cytometry |
Omni-PAGE Hepes-Tris Gels Hepes 4~20%, 10 wells | Epizyme | LK206 | Western blot |
OSCAR(D-19) antibody | Santa Cruz Biotechnology | SC-34235 | Flow cytometry |
PBS (2x) | ZHHC | PW013 | Western blot |
Pentobarbital sodium | Sigma-Aldrich | 57-33-0 | Anesthetization |
Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) | Jacson | 115-035-003 | Western blot |
Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) | Jacson | 111-035-003 | Western blot |
Phosphotungstic acid | RHAWN | 12501-23-4 | Transmission electron microscope |
PKM2(d78a4) xp rabbit mab | Cell Signaling | 4053t | Western blot |
Polyethylene (PE) film | Xiang yi | 200150055 | Transmission electron microscope |
Polyvinylidene fluoride membranes | Roche | 3010040001 | Western blot |
Protease inhibitors | Roche | 4693132001 | Western blot |
Recombinant anti-PGD antibody | Abcam | ab129199 | Western blot |
RIPA lysis buffer | Beyotime | P0013 | Western blot |
SDS-PAGE loading buffer (5x) | Cwbio | CW0027S | Western blot |
Size beads | Invitrogen | F13839 | Flow cytometry |
Tabletop High-Speed Micro Centrifuges | Hitachi | CT15E | EV isolation |
Transmission electron microscope | HITACHI | H-7650 | Transmission electron microscope |
Tween-20 | MP Biomedicals | 19472 | Western blot |
Vortex Mixer Genie | Scientific Industries | SI0425 | EV isolation |
ZetaView BASIC NTA – Nanoparticle Tracking Video Microscope PMX-120 | Particle Metrix | N/A | Nanoparticle tracking analysis |
α-Actinin-4 Rabbit mAb | Abclone | A3379 | Western blot |
β-actin | Cwbio | CW0096M | Western blot |