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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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

Protocol

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.......

Representative Results

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 .......

Discussion

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.......

Acknowledgements

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).

....

Materials

NameCompanyCatalog NumberComments
4% paraformaldehyde Biosharp143174Transmission electron microscope
Alexa fluor 488 anti-goat secondary antibodyYeason34306ES60Flow cytometry
Alexa fluor 488 anti-rabbit secondary antibodyInvitrogenA11008Flow cytometry
Anti-CD18 antibodyAbcamab131044Flow cytometry
Anti-CD81 antibodyAbcamab109201Western blot
anti-CD9 antibodyHuabioET1601-9Western blot
Anti-Mitofilin antibodyAbcamab110329Western blot
APOA1 Rabbit pAbAbcloneA14211Western blot
BCA protein assay kitTIANGENPA115Western blot
BLUeye Prestained Protein LadderSigma-Aldrich94964-500ULWestern blot
Bovine serum albuminMP Biomedical218072801Western blot
Caveolin-1 antibodySanta Cruz Biotechnologysc-53564Western blot
CellMask Orange plasma membrane stainInvitrogenC10045Flow cytometry
ChemiluminescenceAmersham BiosciencesN/AWestern blot
Curved operating scissorJZ Surgical InstrumentJ21040EV isolation
Electronic balanceZhi KeZK-DSTEV isolation
Epoch spectrophotometerBioTekN/AWestern blot
Eppendorf tubesEppendorf3810XEV isolation
Flotillin-1 antibodyPTM BIOPTM-5369Western blot
Gel imaging systemTanon4600Western blot
Golgin84Novusnbp1-83352Western blot
Grids - Formvar/Carbon Coated - Copper 200 meshPolysciences24915Transmission electron microscope
Heparin SolutionStemCell 7980EV isolation
Liberase Research GradeSigma-Aldrich5401127001EV isolation
Microscopic tweezerJZ Surgical InstrumentJD1020EV isolation
NovoCyte flow cytometerACEAN/AFlow cytometry
Omni-PAGE Hepes-Tris Gels Hepes 4~20%, 10 wellsEpizymeLK206Western blot
OSCAR(D-19) antibodySanta Cruz BiotechnologySC-34235Flow cytometry
PBS (2x)ZHHCPW013Western blot
Pentobarbital sodiumSigma-Aldrich57-33-0Anesthetization
Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L)Jacson115-035-003Western blot
Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)Jacson111-035-003Western blot
Phosphotungstic acidRHAWN12501-23-4Transmission electron microscope
PKM2(d78a4) xp rabbit  mab Cell Signaling4053tWestern blot
Polyethylene (PE) filmXiang yi200150055Transmission electron microscope
Polyvinylidene fluoride membranes Roche3010040001Western blot
Protease inhibitorsRoche4693132001Western blot
Recombinant anti-PGD antibodyAbcamab129199Western blot
RIPA lysis bufferBeyotimeP0013Western blot
SDS-PAGE loading buffer (5x)CwbioCW0027SWestern blot
Size beadsInvitrogenF13839Flow cytometry
Tabletop High-Speed Micro CentrifugesHitachiCT15EEV isolation
Transmission electron microscopeHITACHIH-7650Transmission electron microscope
Tween-20MP Biomedicals19472Western blot
Vortex Mixer GenieScientific IndustriesSI0425EV isolation
ZetaView BASIC NTA - Nanoparticle Tracking Video Microscope PMX-120Particle MetrixN/ANanoparticle tracking analysis
α-Actinin-4 Rabbit mAbAbcloneA3379Western blot
β-actinCwbioCW0096MWestern blot

References

  1. Abels, E. R., Breakefield, X. O. Introduction to extracellular vesicles: biogenesis, RNA cargo selection, content, release, and uptake. Cellular and Molecular Neurobiology. 36 (3), 301-312 (2016).
  2. Mathieu, M., Martin-Jaular, L., Lavieu, G., Théry, C.

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Extracellular VesiclesIsolationAnalysisPlasmaSolid TissuesSurface AntigensProtein CargoesFlow CytometryMaxillary BoneLiberaseCentrifugationPBSTissue PreparationSample Processing

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