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* These authors contributed equally
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 vitro
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 take.......
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 .......
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.......
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).
....Name | Company | Catalog Number | Comments |
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 |
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