Cells are known to release different types of vesicles such as small extracellular vesicles (sEVs) and large extracellular vesicles (LEVs). sEVs and LEVs play important roles in intercellular communication, pre-metastatic niche formation, and disease progression; both can be detected cell culture media and biological fluids. sEVs and LEVs contain a variety of protein and RNA cargo, and they are believed to impact many biological functions of the recipient cells upon their internalization or binding to cell surface proteins. It has recently been established that standard isolation techniques, such as differential ultracentrifugation, yield a mixed population of EVs. However, density gradient ultracentrifugation has been reported to allow the isolation of sEVs without cellular debris. Here, we describe the most common methods used to isolate sEVs from cell culture medium, mouse and human plasma, and a new technique for isolating sEVs from tissues as well. This article also provides detailed procedures to isolate LEVs.
Cells are able to communicate with each other through the shedding of extracellular vesicles (EVs), which include small extracellular vesicles (sEVs) and large heterogeneous vesicles[
sEV development starts in multivesicular bodies, which are formed by the intraluminal inward budding of endosomes. These intracellular vesicles frequently fuse with the plasma membrane of the cell, to be released into the extracellular space. LEVs are formed through budding and fission of the plasma membrane[
The size of EVs, including sEVs and LEVs, ranges from a few nanometers to a few micrometers[
To utilize EVs in research, it is important to follow the guidelines proposed by the International Society for Extracellular Vesicles reported in the Minimal Information for Studies of Extracellular Vesicles (MISEV 2018) publication. MISEV 2018 prompts researchers to be careful on their analysis of EVs and provides steps and protocols that can be followed to adequately document the biogenesis, uptake and functions of EVs[
Here, we describe the procedures for successfully isolating sEVs from cell culture medium, plasma or tissues using differential ultracentrifugation and iodixanol density gradients. In addition, we illustrate the isolation of LEVs from cell culture medium using differential ultracentrifugation and iodixanol density gradients. Finally, we discuss the isolation of sEVs using an immunocapture technique.
Laminar flow hood.
Phosphate-buffered saline 1X (PBS).
0.05% trypsin.
Cell culture medium containing
FBS-free cell culture medium.
Sterile cell culture plates.
15-mL and 50-mL conical tubes.
Sterile 10-mL and 25-mL serological pipettes.
Tabletop Eppendorf centrifuge with temperature control (5415R).
Beckman polycarbonate ultracentrifuge tubes, 70-mL (Cat. # 355620), including cap assemblies, for use with 45Ti rotor.
Beckman 45Ti fixed-angle rotor (Cat. # 339160).
Weighing balance.
Beckman L8-70M Ultracentrifuge (for use with 45Ti rotor).
RIPA lysis buffer: 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, pH 8, 1% NP-40, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulfate (SDS). Add protease inhibitor cocktail just prior to cell lysis: PMSF (1 mM), aprotinin (10 µg/mL), leupeptin (10 µg/mL), pepstatin (4 µg/mL), calpain inhibitor (1µM), and sodium orthovanadate (1 mM).
This procedure has been adapted from a previously published protocol, and modifications have been added as needed[
Schematic drawing for extracellular vesicle isolation procedure by differential ultracentrifugation. For extracellular vesicle isolation, the supernatant is collected from cells cultured in dishes for 48 h at 37 °C. In the first step, dead cells and cell debris are spun down from the supernatant at 2000 ×
Culture cells in sterile 150-mm cell culture plates. Grow cells of interest until they reach 70%-80% confluency.
Remove the culture medium and replace it with a similar volume of either FBS-free medium or medium supplemented with sEV-depleted FBS. Use as many plates as necessary to obtain at least 70 mL of conditioned medium.
After an additional 48 h, collect the supernatant. Cells that grow rapidly may become overconfluent and start dying after 48 h. In this case, either collect the supernatant after 24 h or use medium supplemented with sEV-depleted FBS.
Set tabletop centrifuge chamber temperature at 4 °C.
In the laminar flow hood, collect medium in 50-mL conical tubes using a sterile 25-mL serological pipette. Conditioned medium from the same cell line can be combined in tubes.
Place 50-mL tubes in the tabletop centrifuge and centrifuge them for 20 min at 2000 ×
During this spin, perform total cell lysis using RIPA lysis buffer if desired. Store cell lysates at -20 °C or on ice if quantifying proteins and/or analyzing by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
In the laminar flow hood, transfer the supernatant from 50-mL conical tubes to chilled polycarbonate ultracentrifuge tubes. Each tube can be filled with approximately 65 mL of conditioned medium. You can combine supernatants from the same cell line. Discard supernatant from 50-mL conical tubes (leave 2-3 mL at the bottom to ensure the pellet is not disturbed). Note: for collection of sEVs from cell culture medium, the conditioned medium may be kept at 4 °C for up to 1 week, after the first pre-clearing spin. However, you may observe a measurable decrease in sEV protein yield.
Weigh polycarbonate ultracentrifuge tubes on a balance. Equalize ultracentrifuge tube weight with sterile PBS for balance during centrifugation.
Ensure that rubber stoppers are all in place (45Ti rotor), mark one side of each ultracentrifuge tube with a waterproof marker, orient the tube in the rotor with the mark facing up. Centrifuge at 9000 rpm (10,000 ×
Obtain new chilled ultracentrifuge tubes and place them in the laminar flow hood.
In the hood, transfer the supernatant from the centrifuged polycarbonate ultracentrifuge tubes to new polycarbonate ultracentrifuge tubes. Do not disturb or collect any of the pellets, as this material consists of cellular debris and LEVs. Weigh and balance the new tubes with PBS. Spin the supernatants in the ultracentrifuge at 30,000 rpm (100,000 ×
At the end of the spin, aspirate as much supernatant as possible without disturbing the pellet and resuspend all pellets in 40-50 mL of PBS. Pellets from the same cell line may be combined in a single tube. Weigh and if needed balance with a centrifuge tube containing PBS.
Spin in the ultracentrifuge at 30,000 rpm (100,000 ×
Carefully aspirate all supernatant, resuspend the pellet in 100 µL of PBS or iodixanol buffer (see Section 4), aliquot and store at -80 °C until further use. Note: when performing immunoblotting analysis, keep in mind that the level of sEV markers and other proteins of interest may vary in different cell lines. The individual user must determine the amount of protein needed to test for the markers.
1.5-mL Eppendorf tubes.
1× PBS.
Plasma.
Tabletop Eppendorf centrifuge (5415R).
Beckman Coulter 3.5-mL polycarbonate ultracentrifuge tubes (Cat. # 34622).
Beckman TLA 100.3 fixed-angle rotor (Cat. # 349490).
Beckman L8-70M ultracentrifuge (TLA-100.3 fixed-angle rotor).
RIPA lysis buffer: 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, pH 8, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS. Add protease inhibitor cocktail just prior to cell lysis (see Section 2).
This procedure has been adapted from a previously published protocol[
Thaw plasma (3-5 mL) on ice and centrifuge the samples at 1500 ×
Transfer the supernatant to a new Eppendorf tube, and discard the tube containing the pellet. Note: for each tube, collect the supernatant using a pipette and tilt the tube so that the pellet is on the inside of the tube opposite to the supernatant, making sure not to disturb the pellet.
Centrifuge the plasma in the Eppendorf tubes at 12,000 ×
Transfer the plasma to 3.5-mL ultracentrifuge tubes. The remaining pellet contains mostly large vesicles which can be resuspended in PBS for storage or they can be lysed in RIPA lysis buffer if further analysis is desired. Note: this protocol is suitable for human plasma or plasma obtained from animal models, in which 3-5 mL are available. For samples of smaller volume, such as from mice, adjust the volume with clean PBS or use smaller ultracentrifuge tubes and rotors (such as the TLA 100.2) as needed. Alternatively, pool samples from multiple mice. As previously described[
Centrifuge the plasma from step 4 above at 47,000 rpm (110,000 ×
Wash/resuspend each pellet with 1 mL of PBS.
Centrifuge the resuspended pellet at 47,000 rpm (110,000 ×
Remove and discard supernatant. The pellet contains predominantly sEVs. Resuspend the pellet in up to 100 µL of PBS for functional analysis or isolation by iodixanol density gradient ultracentrifugation. Alternatively, they can be lysed with RIPA lysis buffer for immunoblotting or iodixanol buffer to separate sEVs using iodixanol gradients (see Section 5). Note: to concentrate the sEV preparation, resuspend and combine pellets from different preparations of the same sample in 100 µL of PBS. To resuspend the pellet, it is advised to cut the pipette tips to avoid lysing the sEVs. If a pellet is particularly difficult to resuspend, brief exposure (1-3 s) to the tabletop mixer can be used as well.
Store samples at -80 °C or use for SDS-PAGE immediately.
Iodixanol (OptiPrepTM, Sigma # 1556) stock solution (60% wt/vol).
Buffer solution (0.25 M sucrose, 10 mM Tris, pH 8.0, 1 mM EDTA, pH 7.4).
1× PBS.
SETON open-top centrifuge polyallomer tubes.
SW55Ti swinging-bucket rotor.
Beckman L8-70M Ultracentrifuge.
1.5 mL ultra-microfuge tubes.
S55A2 micro-ultracentrifuge rotor.
SorvallTM MTX 150 micro-ultracentrifuge.
ABBE-3L refractometer.
The sEV pellet isolated from cell lines by differential ultracentrifugation (refer to Section 2) can be further isolated using iodixanol density gradient ultracentrifugation. This protocol has been modified from a previously published protocol[
In a 3-4 mL ultracentrifuge tube, prepare a discontinuous gradient of 30%, 20%, and 10% iodixanol solutions:
30% iodixanol (1.6 mL total volume): combine 800 μL of 60% iodixanol/OptiprepTM stock and 800 μL of buffer solution mixed with the sEV pellet;
20% iodixanol (700 μL total volume): combine 233 μL of 60% iodixanol/OptiprepTM stock with 467 μL of buffer solution;
10% iodixanol (700 μL total volume): combine 117 μL of 60% iodixanol/OptiprepTM stock with 583 μL buffer.
Carefully layer 30%, 20%, and 10% gradient solutions (bottom to top) in SETON tubes.
Centrifuge the discontinuous gradient solution at 51,000 rpm (350,000 ×
After the spin, collect 10 fractions of 260 μL each in the 1.5-mL ultra-microfuge tubes starting from the top of the tube.
In separate Eppendorf tubes, collect 10 μL of each fraction to assess their density with an ABBE-3L refractometer and calculate the density using the conversion table provided by the manufacturer (OptiprepTM).
Dilute the 250-μL fractions with 1 mL of PBS. Centrifuge at 100,000 ×
Discard the supernatant and resuspend the pellet with 1 mL of PBS. Centrifuge at 100,000 ×
Resuspend the resulting pellets for each fraction in 30-80 μL of PBS and store them at -80 °C until further use.
Iodixanol (OptiPrepTM, Sigma # 1556) stock solution (60% wt/vol).
Iodixanol buffer solution (0.25 M sucrose, 10 mM Tris, pH 8.0, 1 mM EDTA, pH 7.4)[
1× PBS.
Beckman 13-mL ultracentrifuge tubes.
Beckman SW55Ti swinging-bucket rotor.
Beckman L8-70M Ultracentrifuge.
Beckman TLA-100.2 rotor.
Beckman OptimaTM ultracentrifuge.
ABBE-3L refractometer.
sEVs can be isolated from patient plasma by differential ultracentrifugation (refer to Section 3) and further isolated using iodixanol density gradient ultracentrifugation. This protocol has been modified from a previously published protocol[
Prepare 40%, 20%, 10% and 5% wt/vol iodixanol solutions by diluting a stock solution (60% wt/vol) of iodixanol (OptiPrepTM) with the iodixanol buffer solution.
Mix the sEV pellet with stock iodixanol solution to obtain 783 μL of 40% iodixanol-sEV suspension.
Next, layer the 783 μL of 20% (wt/vol) iodixanol, 783 μL of 10% (wt/vol) iodixanol, and 652 μL of 5% (wt/vol) on top of the 40% iodixanol-sEV suspension to generate a discontinuous iodixanol gradient.
Centrifuge the tubes at 100,000 ×
Starting from the top of the tube, collect 10 fractions of 275 μL each.
Assess the density of each fraction with the ABBE-3L refractometer.
Dilute all fractions and wash them with 1 mL of PBS. Centrifuge the fractions at 100,000 ×
Resuspend the resulting pellets from each fraction in 30 μL of PBS and store them at -80 °C until further use.
The following protocol has been optimized and slightly modified from a previously published protocol[
EDTA buffer: 10 mM EDTA in PBS (pH 8.2).
1.5-mL Eppendorf tubes.
1× PBS.
2-mL ultracentrifuge Eppendorf tubes.
Frozen or fresh tissue.
Eppendorf tabletop centrifuge (5415R).
S55A2 fixed-angle rotor.
SORVALLTM MTX 150 micro-ultracentrifuge.
RIPA lysis buffer: 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, pH 8, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS. Add protease inhibitor cocktail just prior to cell lysis (see Section 2).
Set tabletop centrifuge temperature at 4 °C.
Cut the tissue into small pieces that can fit into the 1.5-mL Eppendorf tubes.
Add 500 μL of EDTA buffer to each tube with tissue pieces.
Incubate tissue pieces for 45 min at room temperature.
After incubation, centrifuge the sample (EDTA buffer + tissue) at 16,000 ×
Transfer the supernatant to 2-mL ultracentrifuge Eppendorf tubes and place the samples in the S55A2 rotor for ultracentrifugation.
Centrifuge the samples at 100,000 ×
Discard the supernatants if the pellet is visible; if the pellet is not visible, leave around 200 μL at the bottom of the tube.
To wash sEVs, add 1 mL of PBS, pipette up and down to resuspend the pellet, and centrifuge the suspension at 100,000 ×
Remove the supernatant and resuspend the pellet in 100 μL of PBS.
Store the vesicles at -80 °C or lyse them with RIPA lysis buffer for immunoblotting to test for sEV markers.
Laminar flow hood.
FBS-free cell culture medium.
Cell culture medium containing
1× PBS.
Sterile cell culture plates.
Sterile 10-mL and 25-mL serological pipettes.
50-mL conical tubes.
Tabletop centrifuge with temperature control for 50-mL conical tubes.
Beckman polycarbonate ultracentrifuge tubes (65 mL, including the cap assemblies, for use with 45Ti rotor).
Beckman 45Ti fixed-angle rotor (Cat. # 339160).
Balance.
Beckman L8-70M ultracentrifuge (for use with the 45Ti rotor).
1.5-mL Eppendorf tubes.
Eppendorf tabletop centrifuge (5415R).
Culture cells of interest in 20 150-mm plates until they reach 70%-80% confluency.
Remove the culture medium and replace it with FBS-free medium; then place the plates in the incubator for 48 h.
After 48 h, collect the medium in 50-mL conical tubes in the hood, using a 25-mL pipette. The medium can be combined in tubes from the same cell lines.
Set the tabletop centrifuge at 4 °C. Centrifuge the 50-mL conical tubes at 2000 ×
Return to the hood and transfer the supernatant from the 50-mL tubes to the prechilled polycarbonate ultracentrifuge tubes. Each tube can be filled with approximately 65 mL of medium. Note: leave approximately 2-3mL of medium at the bottom to avoid disturbing the pellet.
Weigh the polycarbonate ultracentrifuge tubes on the balance. Equalize the weights of tubes for centrifugation by adding sterile PBS to the tubes. Note: ensure that all the rubber stoppers are in place (Beckman 45Ti rotor). Mark one side of each polycarbonate ultracentrifuge tube with a waterproof marker to identify the location of the pellet.
Centrifuge the tubes at 9000 rpm (10,000 ×
Using the 1000-µL pipette, resuspend the pellet in each tube in 1 mL of sterile PBS.
Transfer the LEV suspension to 1.5-mL Eppendorf tubes and label them accordingly.
Centrifuge the Eppendorf tubes in the tabletop centrifuge at 13,200 rpm (16,000 ×
Once the spin is over, return to the hood and aspirate the supernatant without disturbing the pellet.
Resuspend the pellet in each tube in 100 µL of sterile PBS. Combine all the Eppendorf tubes in one tube and label accordingly.
Centrifuge the sample containing the LEVs at 13,200 rpm (16,000 ×
At the end of the spin, carefully aspirate the supernatant, resuspend the LEV pellet in 100 µL of PBS and store at -80 °C for further use.
For isolation of LEVs from cell culture medium via iodixanol density gradient ultracentrifugation, please refer to Section 4, where the LEV pellet (described in step 14) will replace the sEV pellet.
Immunoisolation of sEVs utilizing antibodies that target sEV markers (CD9, CD63 and CD81) or other transmembrane proteins expressed on sEVs, e.g., prostate-specific membrane antigen, offers several advantages. Examples of such advantages are: (1) isolation of concentrated sEV sample; (2) removal of contaminants other than sEVs which may obscure the results; and (3) comparison of potential subpopulations of sEVs. Materials and methods utilized for the immuno-capture of sEVs[
Cell culture- or plasma-derived iodixanol gradient-isolated sEVs (Section 4, 5).
Monoclonal antibodies to tetraspanins (CD9, CD63 and CD81) or to cancer-specific markers present on sEVs (e.g., prostate-specific membrane antigen). Note:
Isotype immunoglobulins.
DynabeadsTM M-270 epoxy beads (Invitrogen, Cat. # 14301).
Microbalance.
1.5-mL Eppendorf tubes.
Buffers (Dynabeads M-270 epoxy beads, Invitrogen, Cat. # 14301): Buffer A: 0.1 M sodium phosphate buffer (pH 7.4) [dissolve 2.62 g NaH2PO4·H2O (MW 137.99) and 14.42 g Na2HPO4·2H2O (MW 177.99) in distilled water, adjust pH if necessary and adjust volume to 1 L]; Buffer B: 3 M ammonium sulfate (stock solution): dissolve 39.6 g (NH4)2SO4 (MW 132.1) in 0.1 M sodium phosphate buffer (pH 7.4) and adjust volume to 100 mL; Buffer C: 0.1 M citrate, pH 3.1: dissolve 2.10 g citric acid (C6H8O7·H2O, MW 210.14) in 90 mL of distilled water, adjust to pH 3.1 and adjust volume to 100 mL.
1× PBS.
Vortex.
DynaMagTM magnet (Invitrogen).
Incubator (37 °C) with tilt rotation.
Mini Mixer (4 °C).
RIPA Lysis buffer: 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, pH 8, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS. Add protease inhibitor cocktail just prior to cell lysis (see Section 2).
The following protocol for immunocapture of sEVs has been optimized and slightly modified from DynabeadsTM M-270 epoxy beads (Invitrogen, Cat. # 14301).
Weigh out 5 mg of lyophilized superparamagnetic Dynabeads (~3.3 × 108 beads) on the microbalance. Put beads in 1.5-mL Eppendorf tubes and resuspend in 1 mL of Buffer A.
Mix well by vortexing for 30 s followed by 10 min incubation at room temperature with tilting and rotating.
Place the Eppendorf tube in the DynaMagTM magnet for 1 min and discard the supernatant.
Remove the tube from the magnet and again resuspend the washed beads in 1 mL of Buffer A; mix well by vortexing for 30 s.
Place the tube on the DynaMagTM magnet for 1 min and discard the supernatant.
Resuspend the washed beads from above in the same volume of Buffer A as calculated for the antibody volume and vortex the suspension. For example, for 5 mg of beads, ~100 µg of antibody is needed. Accordingly, if antibody concentration is 1 mg/mL, a 100-µL antibody volume is needed. Thus, 100 µL of Buffer A is needed for the resuspension of beads.
Add ~100 µL of antibody as in the example above and thoroughly vortex the suspension.
Add the same volume of Buffer B (100 µL as in the example above). Note: with 5 mg of beads in a total of 300 µL of buffer-antibody mix, the coupling concentration is at ~1.1 × 109 beads/mL.
Incubate bead-antibody-buffer mix from step 3 for 16-24 h at 37 °C with slow tilt rotation. Note: beads should not settle during the incubation period.
After the incubation, place the Eppendorf tubes on the DynaMagTM magnet for 2 min, and then remove the supernatant. Note: ensure the collection of any beads adhering to the cap.
Wash the antibody-coated beads four times with 1 mL of PBS, each time resuspending the beads in PBS and applying them to the DynaMagTM magnet for 2 min during each wash.
Resuspend the antibody-coated beads to the desired concentration in PBS (e.g., a volume of 165 µL gives a bead concentration of ~2 × 109 beads/mL).
Add iodixanol gradient-purified sEVs (30-40 µg) to 165 µL of antibody-coupled beads mentioned above.
Incubate overnight with tilting and rotating at 4 °C on a mini mixer to capture the target protein-specific sEVs.
Place the tube on the DynaMagTM magnet for 4 min to collect the bead-sEV complexes at the tube wall. Pipet off the supernatant.
Wash the bead-sEV complexes 3 times using 1 mL of PBS each time by resuspending the beads and applying the DynaMagTM magnet for 2 min during each wash.
The immunocaptured sEVs can either be lysed with RIPA lysis buffer to proceed to immunoblotting analysis or eluted in the following steps.
For elution of sEVs from bead-antibody complex, add 10-100 µL of Buffer C to the beads with immobilized sEVs and mix well by tilting and rotating for 2 min.
Place the Eppendorf tubes on the DynaMagTM magnet for 2 min and transfer the supernatant containing the target protein-specific purified sEVs to a clean tube. Note: change Eppendorf tubes before elution to avoid eluting off non-specific contaminants binding to the tube walls.
The isolation of EVs via differential ultracentrifugation has advantages for users since this technique is cost-effective, yields a large amount of EVs and is relatively easy to learn and perform. Differential ultracentrifugation protocols are well established and readily available to users. This method also has some disadvantages that users need to keep in mind. When isolating EVs through differential ultracentrifugation, co-precipitation of protein aggregates, cellular debris, small non-EV structures and EV-associated RNA can occur; this can lead to decreased sample purity. It should also be noted that the yield of EVs varies depending on the conditions and the cell lines used[
Density gradient isolation allows to preserve the EVs in the gradient by making iso-osmotic solutions at all densities. However, this method may result in low yield of small vesicles and sample loss. Disadvantages associated with density gradient are that larger vesicles can be harder to separate due to their similar sedimentation rates which lead to some inaccuracy in the sample analysis. For functional assays, if using sucrose, it is advised to remove the density gradient medium since it may interfere with some functional assays[
Immunocapture is a very specific technique used to isolate a targeted set of vesicles that are homogeneous and contain similar protein content. This method works efficiently even if the starting material is small. Unfortunately, this technique is not cost-effective and not suited for processing large sample volumes. In addition, bound EVs cannot be used for functional assays since samples will contain antibodies that can affect EV-cell interactions; also eluting the EVs from the beads may not be possible. To isolate the desired population of EVs, it is important to have specific EV markers for each class of EVs[
Additional isolation methods are summarized below although not in detail.
Size-exclusion chromatography (SEC) is now widely used for EV separation. Whether it is through high performance liquid chromatography or gravity-based chromatography at the benchtop, the main principle is as follows. The sample is passed through a column with porous beads made of various materials (predominantly Sepharose). The pore size needs to be small to avoid trapping of the EVs, which will therefore elute from the column sooner than small contaminating proteins such as albumin, which can co-precipitate in other common EV isolation methods such as ultracentrifugation. SEC is also useful for cleaning up antibody or membrane dye-stained EVs, as the labeled sEVs will elute sooner than the unbound dye, qdot, and so on[
Tangential flow filtration (TFF) is a relatively new EV isolation technique known to be able to rapidly process large volumes of cell culture medium or other fluids. It is a fast, efficient and reliable method for isolation of biologically active EVs[
Unlike the traditional dead-end filtration method, where the sample flows perpendicular to the membrane and eventually clogs the membrane pores, in the case of TFF, the fluid flows tangentially across the surface of a semi-permeable membrane with a particular molecular weight cut-off. This causes less clogging and buildup on the membrane[
We describe here the methods that are most commonly used to isolate sEVs from tissues and biological fluids; we also describe the methods that are most commonly used to isolate sEVs and LEVs from cell culture medium. We apologize for not being able to describe other methods for EV isolation or commercially available kits due to space constraints. With a need for transparency and consistency in the field of EV research, investigators are encouraged to report the chosen methods used for isolation and characterization, such as expression of sEV and LEV markers, nanoparticle tracking analysis[
We are grateful to Marja Nevalianen; Carmine Fedele; Andrea Friedman; Jessica Kopenhaver; Huimin Lu for helpful suggestions and to Veronica Robles for help with preparation of the manuscript.
Contributed to this manuscript equally: Salem I, Naranjo NM
Designed, wrote and reviewed the manuscript: Salem I, Naranjo NM, Languino LR
Generated section 3: Sayeed A
Generated sections 2 and 4: Singh A and DeRita R
Generated sections 5 and 8: Krishn SR
Generated section 6: Salem I
Generated section 7: Naranjo NM
Generated
Reviewed and edited the manuscript: Singh A, DeRita R, Krishn SR, Sirman LS, Quaglia F, Duffy A, Bowler N
Not applicable.
Grant support: NIH-CA P01-CA140043 and R01-CA224769. This project is also funded, in part, under a Commonwealth University Research Enhancement Program grant with the Pennsylvania Department of Health (H.R.); the Department specifically disclaims responsibility for any analyses, interpretations or conclusions.
All authors declared that there are no conflicts of interest.
Not applicable.
Not applicable.
© The Author(s) 2020.