Method Article

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

DOI:

10.3791/63990

⸱

October 17th, 2022

 ,  ,  ,  ,  ,  ,  ,  , 
1State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, School of Stomatology, The Fourth Military Medical University, 2Department of Orthodontics, School of Stomatology, The Fourth Military Medical University, 3Department of VIP Dental Care, School of Stomatology, The Fourth Military Medical University, 4School of Basic Medicine, The Fourth Military Medical University, 5College of Life Science, Northwest University, 6Xi'an Institute of Tissue Engineering and Regenerative Medicine

* These authors contributed equally

In This Article

Summary

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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

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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

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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.

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Protocol

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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

  1. Prepare the plasma samples following the steps below.
    1. Determine the body weight of the mouse using a standard laboratory balance.
    2. Add 20 µL, 2 mg/mL of heparin to a 1.5 mL centrifuge tube and use the mixer device (see Table of Materials) to let the heparin attach to the wall of the tube.
      NOTE: Anticoagulation tubes can also be used directly to collect the blood.
    3. Anesthetize the mouse by intraperitoneal injection of 50 mg/kg of pentobarbital sodium (see Table of Materials). Grab the neck of the mouse with the thumb and forefinger, then fix the tail and the left hind leg with the little finger and inject the pentobarbital sodium with a 1 mL injection syringe after exposing the belly.
    4. Confirm that the mouse is properly anesthetized based on the absence of corneal reflex and limb reaction when the footpad is pinched.
    5. Shave the hair on the face and eyelashes of the eyes with curved scissors.
    6. Grab the neck skin of the mouse and press the skin around the eyes to the back of the neck so that the eyeball protrudes. Use ophthalmic tweezers to quickly clamp the eyeball, and collect the outflow of blood with the 1.5 mL centrifuge tubes prepared in step 1.1.2. Sacrifice the mouse by dislocating the cervical vertebra.
      CAUTION: Be careful with the hairs and eyelashes because they may cause hemolysis.
    7. Gently turn the tube upside down several times to prevent blood clotting.
      CAUTION: Turn the tube upside down as soon as possible.
    8. Centrifuge the blood sample for 15 min at 1,200 x g at 4 °C.
    9. Carefully transfer the supernatant to new and clean 1.5 mL centrifuge tubes, and use the plasma sample immediately or store it at 4 °C for a few hours.
    10. Dilute the supernatant with an equal volume of 1x phosphate-buffered saline (PBS) and mix well.
      ​NOTE: The protocol provided above to collect the plasma is fatal. The blood can also be collected through subclavian vein puncture11 without sacrificing the mouse.
  2. Prepare the maxillary bone samples following the steps below.
    1. Isolate the maxillary bone with ophthalmic tweezers and scissors and wash them with PBS to get rid of soft tissues with tweezers.
    2. Put the maxillary bone into 1.5 mL centrifuge tubes and cut the bone into small pieces (the size must be less than 1 mm in diameter) with scissors. Add a certain amount of Liberase (5 µg/mL, see Table of Materials) to cover the tissue (usually 1 mL for bone samples from an 8-week-old mouse), and then incubate for 30 min at 37 °C.
    3. Centrifuge the sample at 800 x g for 10 min at 4 °C.
    4. Carefully transfer the supernatant with a pipette to new and clean 1.5 mL centrifuge tubes.

2. Extraction of EVs

  1. Centrifuge the samples (prepared in step 1.1.10 and step 1.2.4) for 15 min at 2,500 x g (4 °C) to remove large cell debris and remaining platelets.
  2. Carefully transfer the supernatants to new and clean 1.5 mL centrifuge tubes and centrifuge for 30 min at 16,800 x g (4 °C).
    NOTE: The centrifugation speed can be set to over 10,000 x g to extract EVs12. The higher the speed of the centrifuge, the more quantity of EVs can be obtained. Because the speed limit of the centrifuge we used is 16,800 x g (see Table of Materials), we chose this speed in this protocol.
  3. Discard the supernatant and resuspend the pellets in each tube with 1 mL of PBS. Centrifuge for 30 min at 16,800 x g (4 °C).
  4. Discard the supernatant and resuspend the pellets in each tube with 50 µL of PBS. Store these samples at 4 °C for only less than 24 h, or preferably use immediately for the following analyses (steps 3-5).
    ​CAUTION: Storage at -80 °C is unacceptable, as this will reduce the concentration of EVs and increase the particle sizes of EVs13, thus influencing the following analysis of EVs.

3. Morphological identification of EVs

  1. Perform NTA analysis.
    1. According to the amount of EVs (roughly judged by the size of pellets, the particles of 50 µL plasma are the minimum), transfer 5-50 µL of resuspension (step 2.4), dilute in 10 mL of buffer solution (PBS filtered with 0.22 µm filter), and mix with 1 mL micro pipettor tips sufficiently. Use this final suspension for particle measurement.
    2. Use a sterile 1 mL syringe to inject at least 5 mL of distilled water with a moderate and constant speed until the number of particles displayed on the detection interface is less than five.
      NOTE: After injecting 1 mL of distilled water through the whole channel, the number of particles is directly shown on the detection interface. If the number is still over five, continue injecting 1 mL of distilled water until the criteria are met.
    3. Reconstitute 1 µL of calibration solution in 1 mL of distilled water to generate a primary solution, and then take 100 µL of the primary solution to 25 mL of distilled water to prepare a standard stock solution (1:250,000). Store this working reagent at 4 °C for 1 week.
    4. Calibrate the NTA instrument with the standard stock solution (step 3.1.3). Inject 1-5 mL of the working calibration solution with a 1 mL sterile syringe to flush the machine channel until the number of particles displayed on the detection interface is between 50-400 (preferably around 300). Run the calibration program.
    5. Repeat step 3.1.2 to flush the machine channel before each sample measurement.
    6. Use a sterile 1 mL syringe to inject at least 2 mL of buffer solution to flush the machine channel at a constant speed until the number of particles displayed on the detection interface is less than 10.
    7. Inject 1 mL of the EV sample prepared in step 3.1.1 with a moderate and constant speed (the recommended speed is 0.5-1 mL/s).
      NOTE: The optimal particle concentration is making the number of particles displayed on the detection interface range from 50-400, preferably around 300. If the number of particles is too high, repeat step 3.1.2 to flush the machine channel immediately to avoid particle retention at the channel wall and adjust the concentration of the EV sample with step 3.1.1, then repeat from step 3.1.5.
    8. Conduct the particle analysis and generate the analysis reports according to the manufacturer's instructions (see Table of Materials).
    9. If the sample is precious, pump back the EV sample with the 1 mL syringe and collect them in 1.5 mL centrifuge tubes. Repeat step 2.2 to extract the EVs.
    10. After detecting all samples, inject at least 2 mL of buffer solution to the machine channel and then inject at least 5 mL of distilled water until the number of particles displayed on the detection interface is less than five.
    11. Use a sterile 5 mL syringe to inject at least 10 mL of air at a constant speed (the recommended speed is 1 mL/s) to remove the water in the channel.
  2. Perform TEM analysis.
    NOTE: Perform the following steps with fresh EV resuspensions. The formvar-carbon coated electron microscope grid has two sides, with the working side being luminous in the center grids. The minimum volume of plasma to extract the EVs for the below procedure is 50 µL.
    1. Mix the EV sample (step 2.4) with an equal volume of 4% paraformaldehyde (PFA). Deposit 4 µL of the EVs on one grid and incubate for 15 min at room temperature (RT).
    2. Put four drops of PBS (one drop is equal to 50 µL) on a sheet of polyethylene film (see Table of Materials). Transfer the grids with clean microscopic tweezers and wash the working side of the grid in PBS from one drop to another.
    3. Use filter papers to remove extra PBS that remained in the grids.
    4. Incubate the working side of the grid into 1% phosphotungstic acid (see Table of Materials) for 2 min and then repeat step 3.2.2.
      CAUTION: Since phosphotungstic acid is poisonous, this procedure needs to be operated in the fume hood, and redundant phosphotungstic acid needs to be specifically recycled rather than discarded directly.
    5. Put the grids face up in a 10 cm dish covered with filter papers. The grids can be stored at RT for several years.
    6. Observe the grids under an electron microscope following the manufacturer's instructions (see Table of Materials).
      ​NOTE: To ensure the single particles are observed, the concentration of the EVs must be adjusted, and the suspension needs to be clear without obvious turbidity. Before dropping, the sample should be well mixed. The representative TEM and NTA analyses are shown in Figure 2.

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.

  1. Resuspend samples (step 2.4) with 500 µL of PBS and transfer 50 µL into a new and clean 1.5 mL centrifuge tube as a blank control (tube A) and another 50 µL into a new and clean 1.5 mL centrifuge tube as a simple staining tube for FITC (tube B). Add 0.5 µL of membrane dye to the primary tube for membrane staining. Incubate for 5 min at RT.
  2. Centrifuge the primary tube for 30 min at 16,800 x g (4 °C). Discard the supernatant and resuspend the pellets with 200 µL of PBS.
  3. Divide each 50 µL sample into four 1.5 mL centrifuge tubes for simple staining tubes for PE (tube C), surface marker staining (tube D), and secondary antibody only controls (tube E).
  4. Add the primary antibody of OSCAR in bone EV samples as well as CD18 antibody in plasma EV samples (see Table of Materials) to tube B (step 4.1) and tube D (step 4.3) separately (diluted at 1:100). Incubate for 1 h at 4 °C.
  5. Centrifuge the tubes for 30 min at 16,800 x g (4 °C). Discard the supernatant, resuspend the pellets with 500 µL of PBS, and then centrifuge again for 30 min at 16,800 x g (4 °C) to remove the extra primary antibodies.
  6. Discard the supernatant and resuspend the pellets with 50 µL of PBS, followed by adding FITC-conjugated secondary antibodies (see Table of Materials), respectively (diluted at 1:200) (tube E). Add the same secondary antibodies to tube B (step 4.1) and tube D (step 4.3). Incubate all the tubes for 1 h, at 4 °C in the dark.
    NOTE: Direct fluorescence-conjugated labeling antibodies can also be used to process the FC detection with proper isotype controls.
  7. Dilute one drop of 0.2, 0.5, and 1 µm sized beads (see Table of Materials) suspension respectively into 1 mL of PBS, and run each size beads first to make sure of the gate chosen for EVs. Set the threshold of the flow cytometer to search for the beads and EV population by using a suitable forward scatter (FSC) and side scatter (SSC).
    1. Set the terminal condition as calculating 100,000 membrane-dyed particles. Analyze the sample via a flow cytometer under the manufacturer's instructions (see Table of Materials). The representative results are shown in Figure 3.
      ​NOTE: NTA equipment with fluorescence channels can also be used for origin analysis, and the sample preparation is the same as FC.

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.

  1. Resuspend the pellets (step 2.4) in 50 µL of RIPA lysis buffer, and incubate for 30 min on ice.
  2. Quantify the protein concentrations of all samples in the 96-well microplate by the BCA protein assay following the manufacturer's instructions (see Table of Materials).
    1. Dilute the 2 mg/mL of bovine serum albumin (BSA) with 0.9% of normal saline (NS) (containing 0.9% (w/v) of sodium chloride) into 0.5 mg/mL. Add the 0.5 mg/mL of BSA and 0.9% of NS into three duplicated wells with certain volumes, as shown in Table 1.
    2. Drop 2 µL of the samples (step 5.1) and add 18 µL of NS into three duplicated wells separately.
    3. Prepare a working solution by mixing BCA Reagent A with Reagent B (50:1 Reagent A:B). Add 200 µL of the working solution to each well and shake gently for 30 s.
    4. Incubate the 96-well microplate for 20-25 min at 37 °C.
    5. Measure the OD at 596 nm by the spectrophotometer (see Table of Materials).
    6. Export the data.
    7. Draw a standard curve and calculate the protein concentration of the samples.
  3. According to the results, dilute the samples to 1 µg/µL with 0.9% of NS and 5x SDS-PAGE loading buffer (250 mM Tris·HCl, pH 6.8, 10% SDS, 30% (v/v) Glycerol, 10 mM DTT, 0.05% (w/v) Bromophenol blue, see Table of Materials). Seal the tubes with film tightly and heat for 5 min at 100 °C.
  4. Load the samples and the protein ladder into a gradient concentration of 4%-20% Hepes-Tris gel (see Table of Materials).
    NOTE: Choose the gel percentage according to the molecular weight of the proteins of interest.
  5. Run the gel in the running buffer at 80 V until the proteins form a line, and then switch to 120 V for 1 h until the loading dye is at the bottom of the gel.
    NOTE: The run time may vary according to the equipment used or the type and concentration of the gel.
  6. Transfer the gel to the polyvinylidene fluoride (PVDF) membrane (see Table of Materials), pre-incubated in methyl alcohol for 20 s. Use a wet transfer system to transfer for 1 h at 200 mA.
    CAUTION: Ensure that there is no bubble inside the transfer system and keep the PVDF membrane wet.
    NOTE: The transfer time may vary according to the molecular weight of the target protein; 1 KD usually needs 1 min.
  7. Prepare 5% BSA blocking buffer by adding 2.5 g of BSA (see Table of Materials) in 50 mL of Tris-buffered saline-Tween (TBST) (2 mL of Tween added in 2 L of PBS solution) into a 50 mL centrifuge tube. Agitate until the powder is dissolved. Block the membranes in this buffer for 2 h at RT with agitation.
  8. Incubate the membranes with specific primary antibodies diluted with TBST into proper concentration (Mitofilin, 1:1000; α-Actinin-4, 1:1000; CD9, 1:1000; CD81, 1:1000; Apoa1, 1:1000; Golgin84, 1:1000; Flotillin-1, 1:1000; Caveolin-1, 1:1000; PGD, 1:1000; PKM2, 1:1000; β-actin, 1:3000) overnight at 4 °C.
  9. Wash the membranes in the washing buffer (2 mL of Tween added in 2 L of PBS solution) four times (15 min each time), and incubate the membranes with the suitable secondary antibodies at 1:4000 for 1 h at RT with agitation.
  10. Wash the membranes in the washing buffer four times (15 min each time), and image the membranes using the Chemiluminescence Kit and a gel imaging system (see Table of Materials).

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Results

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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 (

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Discussion

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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...

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Disclosures

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The authors have no conflicts of interest to disclose.

Acknowledgements

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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).

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Materials

List of materials used in this article
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

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EV IsolationDifferential CentrifugationPlasma EVsTissue EVsSurface Antigen ProfilingFlow CytometryProtein Cargo AnalysisWestern BlotNanoparticle Tracking

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