This work was supported by NCI grant nos. U54CA143803, CA163124, CA093900, and CA143055 to K. J. P. This research was supported by a grant of the Korea Health Technology R&#38;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &#38; Welfare, Republic of Korea (grant number: HI19C1122). Work by J. Kim and Y.-K. Cho was supported by Institute for Basic Science (IBS-R020-D1), funded by the Korean Government. The authors thank the current and past members of the Brady Urological Institute, especially members of the Pienta-Amend laboratory, for the critical reading of the manuscript.

1. EV isolation and on-chip immuno-fluorescent EV labeling
<ol>
	<li>Collection of cell culture media (CCM) and pre-processing of CCM for EV isolation
	<ol>
		<li>Seed PC3 cells at 30% confluency in a 75 cm2 cell culture flask. Allow control cells to grow to 90% confluency (~48 h) in standard media and cell-line-specific supplements. 
		NOTE: To prevent EV-containing components from affecting cellular uptake (i.e., fetal bovine serum), use exosome-depleted media and supplements.</li>
		<li>Harvest the CCM.</li>
		<li>Centrifuge the CCM at 1000 x g for 10 min at room temperature (RT) to pellet any unattached cells and large debris harvested with the media. Transfer the supernatant to a new conical tube.</li>
		<li>In the new tube, centrifuge the supernatant at 10,000 x g for 20 min at 4 &#176;C to pellet smaller debris and apoptotic bodies remaining in the media. Some larger EVs will pellet. Transfer the supernatant to a new tube.</li>
		<li>Filter the supernatant through a 0.45 &#181;m hydrophilic Polyvinylidene fluoride (PVDF) membrane syringe filter. 
		NOTE: If not immediately processing CCM for EV isolation, store the pre-processed CCM at -80 &#176;C until isolation is performed. If frozen, limit freeze-thaw cycles to one.</li>
	</ol>
	</li>
	<li>EV isolation from CCM using a nano-filtration based microfluidic device
	<ol>
		<li>If frozen, completely thaw CCM and vortex for 30 s before step 1.2.2.</li>
		<li>Inject 1 mL of pre-processed CCM (step 1.1) into the sample chamber of the nano-filtration-based microfluidic device (see Table of Materials)17,26. 
		NOTE: Follow the standard operating procedure for the nano-filtration-based microfluidic device17,26.</li>
		<li>Spin at 3000 rpm for 10 min in the bench-top spinning machine (see Table of Materials) to operate the microfluidic device. 
		NOTE: If CCM remains on the sample chamber following the initial run, perform additional spins until all CCM has emptied from the sample chamber.</li>
		<li>Remove the fluid from the waste chamber by pipetting and repeat steps 1.2.1, 1.2.2, and 1.2.3 twice. 
		NOTE: In total, 3 mL of CCM will be processed for EV isolation.</li>
		<li>Inject 1 mL phosphate-buffered saline (PBS) into the sample chamber to wash the isolated EVs. Spin in the bench-top spinning machine for operating the microfluidic device as mentioned in step 1.2.3. Locate the pure EVs on the membrane of the device. 
		NOTE: The quality of EVs isolated from the nano-filtration based microfluidic device, specifically, was confirmed and compared to the conventional UC method by transmission electron microscopy (TEM), scanning electron microscope (SEM), nanoparticle tracking analysis (NTA), structured illumination microscopy, enzyme-linked immunosorbent assay and real-time PCR in the previous research17,26.</li>
	</ol>
	</li>
	<li>Immunofluorescent labeling of EV using nano-filtration based microfluidic device (Figure 1)
	<ol>
		<li>Select an EV-specific antibody according to the purpose of the assay (see Table of Materials). 
		NOTE: Certain antibodies may interfere with ligand binding sites specific to EV-uptake pathways (i.e., endocytosis).</li>
		<li>Inject 1 &#181;g/mL of the EV-specific antibody into the elution hole of the device containing 100 &#181;L of isolated EVs.</li>
		<li>Incubate for 1 h in the dark at RT on a plate shaker to ensure the even distribution of the antibody across the sample.</li>
		<li>Attach an adhesive tape to the elution hole. Inject 1 mL PBS into the sample chamber to wash out any residual antibodies.</li>
		<li>Spin the device at 3000 rpm until the sample chamber is empty. Remove any fluid from the waste chamber by pipetting. Inject 1 mL PBS into the sample chamber. 
		NOTE: Fluorescently labeled EVs will be located in the membrane chamber.</li>
		<li>Pipette the fluorescently labeled EVs (Figure 2) from the membrane chamber to an amber tube. Block from light until use.</li>
	</ol>
	</li>
</ol>
2. Incubation of the cells with fluorescently labeled EVs for the EV-uptake assay
<ol>
	<li>Target cell seeding and culture on the cell-culture compatible dishes.
	<ol>
		<li>Seed 1 x 104 PC3 cells into the microslide 8-well plate (9.4 x 10.7 mm for each well) with 0.2 mL of media or 4 x 104 PC3 cells into a 35 mm dish with 1 mL of media. Plate the cells into a cell-culture compatible dish consisting of a thin coverslip (thickness: 0.18 mm). 
		NOTE: The thin coverslip minimizes the adverse scattering of light.</li>
		<li>Allow cells to adhere overnight in optimal cell-culture conditions (37 &#176;C, 5% CO2 concentration, 90% humidity).</li>
		<li>Wash adhered cells twice with exosome-depleted media (described in step 1.1.1).</li>
	</ol>
	</li>
	<li>Cell incubation with fluorescently labeled EVs.
	<ol>
		<li>Measure the concentration of fluorescently labeled EVs (step 1.3.5) by nanoparticle tracking analysis (NTA, Supplementary Figure 1). Determine the optimal concentration of fluorescently labeled EVs to be added to the cultured cells (step 2.1.2.).</li>
		<li>Dilute the fluorescently labeled EVs with exosome-depleted media to match the desired concentration measured in step 2.2.1. (i.e., 7.80 x 109 EVs (in NTA value) in 200 &#181;L of exosome-depleted media.)</li>
		<li>Add the diluted EVs (Step 2.2.2) to the adhered target cells prepared at 2.1.2. Incubate for experimental time (i.e., 4, 8, or 12 h).</li>
		<li>Wash cells thrice with exosome-free media to remove any non-internalized EVs. 
		NOTE: Optional: Cells can be fixated following wash.</li>
		<li>Label the cytoplasm of the adhered cells with 1 &#181;g/mL of CMTMR ((5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine) (see Table of Materials) and incubate in optimal cell-culture conditions (37 &#176;C, 5% CO2 concentration, 90% humidity). 
		NOTE: Cell area dyes should fluoresce separately from labeled EVs to aid in determining the spatial location (internalized or superficial) of the spiked EVs during the EV uptake assay.</li>
		<li>Wash labeled cells twice with exosome-depleted media to remove the residual dye. Add fresh exosome-depleted media to the cells in preparation for live-cell confocal imaging.</li>
	</ol>
	</li>
</ol>
3. Confocal microscopy
<ol>
	<li>To perform live-cell imaging, utilize an on-stage incubator to maintain optimal cell-culture conditions (37 &#176;C, 5% CO2 concentration, 90% humidity).</li>
	<li>Place the prepared cells in the on-stage incubator.</li>
	<li>Set the imaging parameters based on control samples. 
	NOTE: Suggested control samples include: Fluorescently labeled EVs only, fluorescently labeled cells, unlabeled EVs, and unlabeled cells.</li>
	<li>Determine the depth of the target cells and the range of stacking size in the z-direction to acquire 3D confocal images. 
	NOTE: The thickness of a Z-stack is 1 &#181;m. The confocal 3D image acquisition lasted 2 min 34 s (each Z-plane image acquisition took approximately 8 s; a total of twenty Z-stack images).</li>
	<li>Set image acquisition to multiple z-stacked images of both cell-specific dye (i.e., red) and EV-specific dye (i.e., green) simultaneously (Figure 3 and Figure 4A).</li>
</ol>
4. Image processing
<ol>
	<li>Utilize automatic image-processing software to analyze the raw z-stacked confocal images and determine the EV uptake by cells (see Table of Materials).</li>
	<li>Set thresholding parameters to the fluorescent signal of the cells and EV-specific dyes. Build the virtual surfaces of cells (Figure 4A,B).
	<ol>
		<li>To build the virtual surfaces of cells, click the button Add new Surfaces.</li>
		<li>Select Shortest Distance Calculation as &#34;Algorithm Settings&#34; to use the provided algorithm by the software, then click Next: Source Channel.</li>
		<li>Select Channel 2 - CMTMR as &#34;Source Channel&#34; in this experiment.</li>
		<li>Select Smooth and put the appropriate value into &#34;Surfaces Detail&#34; for surface smoothing. 
		NOTE: 0.57 &#181;m in this experiment since 1 pixel represents 0.57 &#181;m in raw imaging data.</li>
		<li>Select Absolute Intensity as &#34;Thresholding.&#34;</li>
		<li>To automatically threshold the fluorescent image by the provided algorithm, click Threshold (Absolute Intensity): The value is automatically set.</li>
		<li>Select Enable as &#34;Split touching Objects (Region Growing)&#34; and put the value of estimated cell size into &#34;Seed Points Diameter,&#34; 10.0 &#181;m in this experiment. Then click Next: Filter Seed Points.</li>
		<li>To configure the virtual cell surfaces, click + Add button, then select Quality as &#34;Filter Type.&#34; Threshold the appropriate value (210 in this experiment) for the low limit by a visual inspection and the maximum value (1485) for the upper limit, then click the Finish button. 
		NOTE: A visual inspection means that a researcher can discriminate the cellular area from a raw fluorescent image.</li>
		<li>Next, to build the virtual dots of EVs, click the button Add new Spots.</li>
		<li>Select Different Spot Sizes (Region Growing) and Shortest Distance Calculation as &#34;Algorithm Settings,&#34; then click Next: Source Channel.</li>
		<li>Select Channel 1 - Alexa Fluor 488 as &#34;Source Channel&#34; in this experiment.</li>
		<li>Put the appropriate value into &#34;Estimated XY Diameter&#34; for the spot detection, 1 &#181;m in this experiment. Then, click Next: Filter Spots.</li>
		<li>To configure the virtual EV dots, click + Add button, select Quality as &#34;Filter Type,&#34; and set &#34;Lower Threshold&#34; by a visual inspection, 100 in this experiment. Then, click the Next: Spot Region Type button.</li>
		<li>Select Absolute Intensity as &#34;Spot Regions Type,&#34; then click Next: Spot Regions.</li>
		<li>To threshold, the region of EV dots, put the appropriate value into &#34;Region Threshold&#34; by a visual inspection, 100 as &#34;Region Threshold&#34; in this experiment. 
		NOTE: A visual inspection means that a researcher can discriminate the EVs area from a raw fluorescent image.</li>
		<li>Select Region Volume as &#34;Diameter from,&#34; then click Finish.</li>
	</ol>
	</li>
	<li>Use the software&#39;s provided algorithms to split the grouped spots inside the built surface at step 4.2 (Figure 4C, i-iv).
	<ol>
		<li>Click the built Spots, then go into Filters.</li>
		<li>Click + Add button, then select Shortest Distance to Surfaces Surfaces = Surface 1 as Filter Type, then click Duplicate Selection to new Spots button. The lowest threshold (-7.0 in this experiment) for the low limit and the appropriate value (-0.5) for the upper limit. 
		NOTE: Set the upper limit with the estimated radius of Spots. In this experiment, the estimated diameter of Spots,i.e., EV dots, was set to 1 &#181;m in step 4.2.11.; thus, the upper limit can be 0.5.</li>
	</ol>
	</li>
	<li>Automatic count of EVs inside the cells 
	NOTE: The software will automatically count the number of EVs inside the cells, indicating the number internalized by the target cells.
	<ol>
		<li>Click the built Spots 1 selection [Shortest Distance to Surfaces Surfaces = Surfaces 1 between -7.00 and -0.500].</li>
		<li>Go to the Statistics, and export the value from &#34;Total Number of Spots&#34; 
		NOTE: The software&#39;s provided algorithms will automatically calculate the number and volume of cells.</li>
	</ol>
	</li>
	<li>Determine the yield of EV uptake per incubation period based on the above-calculated values (Figure 5).
	<ol>
		<li>To obtain the number of cells, click the built Surfaces 1, then go to the Statistics, and export the value of &#34;Total Number of Surfaces&#34; from Overall.</li>
		<li>Go to the Detailed into Statistics to export the Volume from Detailed.</li>
	</ol>
	</li>
</ol>

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Y.-K. Cho is an inventor of the patents on the nano-filtration-based microfluidic device, Exodisc, which are licensed to Labspinner (Ulsan, Korea). All other authors have nothing to disclose.

An EV uptake assay based on 3D fluorescence imaging via confocal microscopy provides an efficient methodology and sensitive analysis. This fluorescent EV labeling facilitates the visualization of EVs and successfully performs a precise EV uptake assay. Previous methods for labeling EVs and removing the residual dye have been reported by removing precipitation using ultracentrifugation (UC); however, UC may co-precipitate EVs, and the immobilized dye may lead to a false-positive signal12<s...

Extracellular vesicles (EVs) are nano-sized, lipid membrane-bound particles that are categorized by their sizes: ectosomes (100-500 nm) and exosomes (50-150 nm)1. EVs contain various biomolecules, such as proteins, nucleic acids, and lipids. These biomolecules originate from the cells before being encapsulated as cargo and released into the extracellular space via EVs1,2,3.
Due to the variety of their cargo, EVs are believed to play an active role in intercellular communication. The release and uptake of EVs by cells allow the transfer of biomolecules between the cells4,5. The introduction of EV cargo to a cell may alter the recipient cell&#39;s functions and homeostatic state4,5,6. EVs are internalized through multiple pathways; however, the exact mechanisms have not been accurately demonstrated.
The majority of the EV uptake assays, such as genetic tagging, fluorescently label individual EVs7. The resulting signal can be measured by microplate photometer, flow cytometry, or microscopy, with each technology having substantial limitations. Microplate photometers, flow cytometry, or standard two-dimensional (2D) microscopy cannot distinguish between internalized and superficially attached EVs8,9. Additionally, the necessary sample preparation for each of these techniques may introduce additional issues to EV uptake evaluation. For example, lifting adhered cells with trypsin before EV uptake analysis may cleave some superficially attached EVs on the cell&#39;s surface10,11. Trypsin may also interact with the cell surface, affecting cell and EV phenotype. Additionally, trypsin may not detach superficial EVs entirely, skewing isolated populations.
To accurately label EVs with fluorescent dyes, additional wash steps are required to remove the residual dye7. Accepted isolation techniques can also contribute to false-positive signals due to coagulation that occurs during EV isolation. For example, serial ultracentrifugation (UC) is widely used to isolate EVs and remove the immobilized dye. However, UC may co-precipitate EVs, and the residual dye may lead to a false-positive signal12,13. Other nano-filtration methods, such as column-based filtration, are also widely used for non-immobilized dye removal. The complex nature of EVs and dye interacting within the column matrix may lead to incomplete removal of residual dye due to the molecular cut-off of the column being altered by the complex input14,15,16.
The current protocol proposes a nano-filtration-based microfluidic device to isolate and wash fluorescently labeled isolated EVs. The nano-filtration-based microfluidic device can provide efficient filtration via fluid-assisted separation technology (FAST)17,18. FAST reduces the pressure drop across the filter, thus reducing potential aggregation between EVs and dyes. By efficiently removing residual dye, it is possible to enhance the quality of fluorescently labeled EVs and the assay&#39;s specificity.
Confocal microscopy can distinguish between internalized and superficially attached EVs on the cell surface and comprehensively investigate the cellular mechanisms of EV uptake in a spatiotemporal resolution19,20,21,22,23,24,25. For example, Sung et al. described the visualization of the exosome lifecycle using their developed live-cell reporter. The location of the internalized EVs was detected and analyzed using a confocal microscope in three-dimension (3D) and post-image processing tools20. Although the size of small EVs (40-200 nm) is below the resolution limit of the optical microscope, the fluorescently labeled EVs can be detected by confocal microscopy since the photodetector can detect the enhanced fluorescence emission. Therefore, the subcellular localization of the fluorescently labeled EVs within a cell can be precisely determined by acquiring multiple z-stacked images of the EVs and the surrounding cellular organelles.
Additionally, 3D reconstruction and post-data processing can provide further insight into the positioning of the internalized, superficial, and free-floating EVs. By utilizing these processes in conjunction with the time-lapse live-cell imaging offered by confocal microscopy, the level of EV uptake can be precisely evaluated, and the real-time tracking of EV uptake is also possible. Further, EV trafficking analysis can be performed using confocal microscopy by assessing the co-localization of EVs with organelles, a first step to determine how internalized EVs are involved in the intracellular function. This protocol describes the methodology for performing an EV uptake assay using the nano-filtration-based microfluidic device17,26, confocal microscopy, and post-image analysis.

<table><tbody><tr><td>Alexa Fluor 488 anti-human CD63 Antibody</td><td>Biolegend</td><td>353038</td><td>Fluorescent dye conjugated EV-specific antibody</td></tr><tr><td>CellTracker Orange CMTMR Dye</td><td>Thermo Fisher Scientific</td><td>C2927</td><td>Live cell (cytoplasm) fluoresent labeling reagent</td></tr><tr><td>CFI Apo Lambda S 40XC WI</td><td>Nikon</td><td>MRD77400</td><td>Objective for confocal imaging, NA=1.25</td></tr><tr><td>CFI Plan Apo VC 20X</td><td>Nikon</td><td>MRD70200</td><td>Objective for confocal imaging, NA=0.75</td></tr><tr><td>Exodisc</td><td>Labspinner Inc.</td><td>EX-D1001</td><td>A nano-filtration based microfluidic device for EV isolation</td></tr><tr><td>ExoDiscovery</td><td>Labspinner Inc.</td><td>EX-R1001</td><td>Operation device for Exodisc</td></tr><tr><td>Exosome-depleted FBS</td><td>Thermo Fisher Scientific</td><td>A2720801</td><td>Nutrient of cell culture media for PC3 cell line derived EV collection</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>VWR</td><td>1500-500</td><td>Nutrient for cell cultivation</td></tr><tr><td>Goat Anti-Mouse IgG H&amp;amp;L preadsorbed</td><td>abcam</td><td>&amp;nbsp;ab7063</td><td>Mouse IgG antibody for negatvie control of EV labeling</td></tr><tr><td>Ibidi USA&amp;nbsp;U DISH &amp;mu;-Dish 35 mm</td><td>Ibidi</td><td>81156</td><td>Culture dish for confocal imaging</td></tr><tr><td>Imaris 9.7.1</td><td>Oxford Instruments</td><td>9.7.1</td><td>Post-image processing software</td></tr><tr><td>Incubator System+ CO&lt;sub&gt;2&lt;/sub&gt;/O&lt;sub&gt;2&lt;/sub&gt;/N&lt;sub&gt;2&lt;/sub&gt; gas mixer</td><td>Live Cell Instrument</td><td>TU-O-20</td><td>Incubator system for live cell imaging</td></tr><tr><td>Nikon A1 HD25 / A1R HD25 camera</td><td>Nikon</td><td>NA</td><td>Camera for confocal imaging</td></tr><tr><td>Nikon Eclipse Ti microscope</td><td>Nikon</td><td>NA</td><td>Inverted microscope for confocal imaging</td></tr><tr><td>NIS-Elements AR 4.50.00</td><td>Nikon</td><td>4.50.00</td><td>Image processing software for Nikon microscope</td></tr><tr><td>NTA, NanoSight NS500</td><td>Malvern Panalytical</td><td>NS500</td><td>Measurement device for EV concentration</td></tr><tr><td>OriginPro 2020</td><td>OriginLab</td><td>9.7.0.185</td><td>Graphing software</td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>Antibiotics for cell cultivation</td></tr><tr><td>RPMI 1640</td><td>Thermo Fisher Scientific</td><td>21875034</td><td>Cell culture media for PC3 cell line cultivation</td></tr><tr><td>SYTO RNASelect Green Fluorescent cell Stain - 5 mM Solution in DMSO</td><td>Thermo Fisher Scientific</td><td>S32703</td><td>RNA staining fluorescent dye for the EV labeling</td></tr></tbody></table>

extracellular vesicle uptake assay via confocal microscope imaging analysis

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular communication in various research fields; cancer biology, neuroscience, and drug delivery. Many EV uptake assays have been reported in the literature; however, there is a lack of practical, detailed experimental methodology. EV uptake can be assessed by fluorescently labeling EVs to detect their location within cells. Distinguishing between internalized EVs in cells and the superficial EVs on cells is difficult, yet critical, to accurately determine the EV uptake. Therefore, an assay that efficiently quantifies EV uptake through three-dimensional (3D) fluorescence confocal microscopy is proposed in this work. Fluorescently labeled EVs were prepared using a nano-filtration-based microfluidic device, visualized by 3D confocal microscopy, and then analyzed through advanced image-processing software. The protocol provides a robust methodology for analyzing EVs on a cellular level and a practical approach for efficient analysis.

Using a nano-filtration-based microfluidic device, EVs were isolated from PC3 CCM and labeled with a fluorophore-conjugated EV-specific (CD63) antibody (Figure 1). The labeled EVs were successfully visualized by the 3D confocal microscopy (Figure 2). The labeled EVs were incubated with cells for several hours in exosome-depleted media. Following incubation, cells were washed with exosome depleted media. The remaining EVs were internalized or adhered to cells dur...

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Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Extracellular vesicles (EVs) contribute to cellular biology and intercellular communications. There is a need for practical assays to visualize and quantify EVs uptake by the cells. The current protocol proposes the EV uptake assay by utilizing three-dimensional fluorescence imaging via confocal microscopy, following EV isolation by a nano-filtration-based microfluidic device.

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular ...

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7.4K Views.  Johns Hopkins University School of Medicine. Dit protocol biedt een robuuste methodologie voor het analyseren van EV's op cellulair niveau en een praktische aanpak voor efficiënte EV-opname en EV-trackinganalyse. Deze methode van EV-etikettering elimineert co-neerslag van EV's en kleurstof, waardoor de EV-signalen worden verbeterd. Ev-opname wordt gemeten door volumetrische analyse om de geïnternaliseerde EV's te onderscheiden van de oppervlakkige EV's.EV's zijn actieve lading, dus hun opname maakt de overdracht van biochemisch...

Video: Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released from many cell types and play a pivotal role in regulating cell-cell communication and a diverse range of biological processes. Many different methods for the characterization of EVs have been described. However, most of these methods have the disadvantage that the preparation and characterization of the samples are very time-consuming, or it is extremely difficult to analyze specific markers of interest due to their small size and due to the lack of discrete populations. While methods for analysis of EVs have been considerably improved over the last decade, there is still no standardized method for characterization of single EVs. Here, we demonstrate a semi-automated method for characterization of single EVs by fluorescence-based nanoparticle-tracking analysis. The protocol that is presented addresses the common problem of many researchers in this field and provides the complete workflow for rapid isolation of EVs and characterization with PKH67, a general cell membrane linker, as well as with specific surface markers such as CD63, CD9, vimentin, and lysosomal-associated membrane protein 1 (LAMP-1). The presented results show a high level of reproducibility, as confirmed by other methods, such as Western blotting. In the conducted experiments, we exclusively used EVs isolated from human serum samples, but this method is also suitable for plasma or other body fluids and can be adjusted for characterization of EVs from cell culture supernatants. Irrespective of the future progress of research on EV biology, the protocol that is presented here provides a rapid and reliable method for rapid characterization of single EVs with specific markers.

In this protocol, we describe the complete workflow for rapid isolation of extracellular vesicles from human whole blood and characterization of specific markers by fluorescence-based nanoparticle-tracking analysis. The presented results show a high level of reproducibility and can be adjusted to cell culture supernatants.

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released ...

Biochemistry

Evaluation of Extracellular Vesicle Function During Malaria Infection

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible for most malaria-related deaths globally. Several factors including parasite sequestration in tissues, vascular dysfunction, and inflammatory responses influence the evolution of the disease in malaria-infected people. P. falciparum-infected red blood cells (iRBCs) release small extracellular vesicles (EVs) containing different kinds of cargo molecules that mediate pathogenesis and cellular communication between parasites and host. EVs are efficiently taken up by cells in which they modulate their function. Here we discuss strategies to address the role of EVs in parasite-host interactions. First, we describe a straightforward method for labeling and tracking EV internalization by endothelial cells, using a green cell linker dye. Second, we report a simple way to measure permeability across an endothelial cell monolayer by using a fluorescently labeled dextran. Finally, we show how to investigate the role of small non-coding RNA molecules in endothelial cell function.

In this work, we describe protocols to investigate the role of extracellular vesicles (EVs) released by Plasmodium falciparum infected erythrocytes. In particular, we focus on the interactions of EVs with endothelial cells.

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

Immunology and Infection

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven&#160;OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 &#181;m sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their ...

Bioengineering

Uptake of Fluorescent Labeled Small Extracellular Vesicles In Vitro and in Spinal Cord

Small extracellular vesicles (sEVs) are 50-150 nm vesicles secreted by all cells and present in bodily fluids. sEVs transfer biomolecules such as RNA, proteins, and lipids from donor to acceptor cells, making them key signaling mediators between cells. In the central nervous system (CNS), sEVs can mediate intercellular signaling, including neuroimmune interactions. sEV functions can be studied by tracking the uptake of labeled sEVs in recipient cells both in vitro and in vivo. This paper describes the labeling of sEVs from the conditioned media of RAW 264.7 macrophage cells using a PKH membrane dye. It shows the uptake of different concentrations of labeled sEVs at multiple time points by Neuro-2a cells and primary astrocytes in vitro. Also shown is the uptake of sEVs delivered intrathecally in mouse spinal cord neurons, astrocytes, and microglia visualized by confocal microscopy. The representative results demonstrate time-dependent variation in the uptake of sEVs by different cells, which can help confirm successful sEVs delivery into the spinal cord.

Uptake of Fluorescent Labeled Small Extracellular Vesicles In Vitro and in Spinal Cord

We describe a protocol to label macrophage-derived small extracellular vesicles with PKH dyes and observe their uptake in vitro and in the spinal cord after intrathecal delivery.

Small extracellular vesicles (sEVs) are 50-150 nm vesicles secreted by all cells and present in bodily fluids. sEVs transfer biomolecules such as RNA, ...

Neuroscience

Size Determination and Phenotypic Analysis of Urinary Extracellular Vesicles using Flow Cytometry

Extracellular vesicles, EVs, are a heterogeneous complex of lipidic membranes, secreted by any cell type, in any fluid such as urine. EVs can be of different sizes ranging from 40-100 nm in diameter such as in exosomes to 100-1000 nm in microvesicles. They can also contain different molecules that can be used as biomarkers for the prognosis and diagnosis of many diseases. Many techniques have been developed to characterize these vesicles. One of these is flow cytometry. However, there are no existing reports to show how to quantify the concentration of EVs and differentiate them by size, along with biomarker detection. This work aims to describe a procedure for the isolation, quantification, and phenotypification of urinary extracellular vesicles, uEVs, using a conventional cytometer for the analysis without any modification to its configuration. The method&#39;s limitations include staining a maximum of four different biomarkers per sample. The method is also limited by the amount of EVs available in the sample. Despite these limitations, with this protocol and its subsequent analysis, we can obtain more information on the enrichment of EVs markers and the abundance of these vesicles present in urine samples, in diseases involving kidney and brain damage.

This protocol describes a method for the isolation of urinary extracellular vesicles, uEVs, from healthy human donors and their phenotypic characterization by the size and surface marker expression using flow cytometry.

Extracellular vesicles, EVs, are a heterogeneous complex of lipidic membranes, secreted by any cell type, in any fluid such as urine. EVs can be of ...

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often require indirect methods due to their small diameter (40-250 nm), which is beneath the diffraction limit of typical light microscopy. We have developed a super-resolution microscopy-based visualization of EVs to bypass the diffraction limit in both two and three dimensions. Using this approach, we can resolve the three-dimensional shape of EVs to within +/- 20 nm resolution on the XY-axis and +/- 50 nm resolution along the Z-axis. In conclusion, we propose that super-resolution microscopy be considered as a characterization method of EVs, including exosomes, as well as enveloped viruses.

Direct stochastic optical reconstruction microscopy (dSTORM) is used to bypass the typical diffraction limit of light microscopy and to view exosomes at the nanometer scale. It can be employed in both two and three dimensions to characterize exosomes.

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often ...

Single-Particle Interferometric Reflectance Imaging Characterization of Individual Extracellular Vesicles and Population Dynamics

Extracellular vesicles (EVs) are nanometer-sized vesicles with a lipid bilayer that are secreted by most cells. EVs carry a multitude of different biological molecules, including protein, lipid, DNA, and RNA, and are postulated to facilitate cell-to-cell communication in diverse tissues and organs. Recently, EVs have attracted significant attention as biomarkers for diagnostics and therapeutic agents for various diseases. Many methods have been developed for EV characterization. However, current methods for EV analysis all have different limitations. Thus, developing efficient and effective methods for EV isolation and characterization remains one of the crucial steps for this cutting-edge research field as it matures. Here, we provide a detailed protocol outlining a single-particle interferometric reflectance imaging sensor (SP-IRIS), as a method that is capable of detecting and characterizing EVs from unpurified biological sources and purified EVs by other methodologies. This advanced technique can be used for multi-level and comprehensive measurements for the analysis of EV size, EV count, EV phenotype, and biomarker colocalization.

This protocol presents single-particle interferometric reflectance imaging that is designed for the multi-level and comprehensive measurements of extracellular vesicles (EV) size, EV count, EV phenotype, and EV biomarker colocalization.

Extracellular vesicles (EVs) are nanometer-sized vesicles with a lipid bilayer that are secreted by most cells. EVs carry a multitude of different ...

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and first-generation particle analysis to comprehensive multi-parameter measurements such as nano-flow cytometry (nFCM). nFCM is a form of flow cytometry that utilizes instrumentation specifically designed for nano-particle analysis, allowing for thousands of EVs to be characterized per minute both with and without the use of staining techniques. High resolution side scatter (SS) detection allows for size and concentration to be determined for all biological particles larger than 45 nm, while simultaneous fluorescence (FL) detection identifies the presence of labeled markers and targets of interest. Labeled subpopulations can then be described in quantitative units of particles/mL or as a percentage of the total particles identified by side scatter.
Here, EVs derived from conditioned cell culture media (CCM) are labeled with both a lipid dye, to identify particles with a membrane, and antibodies specific for CD9, CD63, and CD81 as common EV markers. Measurements of comparison material, a concentration standard and a size standard of silica nanospheres, as well as labeled sample material are analyzed in a 1-minute analysis. The software is then used to measure the concentration and size distribution profile of all particles, independent of labeling, before determining the particles that are positive for each of the labels.
Simultaneous SS and FL detection can be utilized flexibly with many different EV sources and labeling targets, both external and internal, describing EV samples in a comprehensive and quantitative manner.

The latest generation of EV characterization tools are capable of single EV analysis across multiple parameters simultaneously. Nano-flow cytometry measures all biological particles larger than 45 nm without labeling and identifies specific characteristics of subpopulations by a variety of fluorescent labeling techniques.

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and ...

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter and are used as a means of intercellular communication. They are emerging as promising diagnostic and therapeutic tools for a variety of diseases. There are two main biogenesis processes used by cells to produce EVs with differences in size, composition, and content. Due to their high complexity in size, composition, and cell origin, their characterization requires a combination of analytical techniques. This project involves the development of a new generation of multiparametric analytical platforms with increased throughput for the characterization of subpopulations of EVs. To achieve this goal, the work starts from the nanobioanalytical platform (NBA) established by the group, which allows an original investigation of EVs based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy (AFM) of vesicular targets trapped on a microarray biochip. The objective was to complete this EV investigation with a phenotypic and molecular analysis by Raman spectroscopy. These developments enable the proposal of a multimodal and easy-to-use analytical solution for the discrimination of EV subsets in biological fluids with clinical potential

This paper proposes a new generation of multiparametric analytical platforms with increased throughput for the characterization of extracellular vesicle subsets. The method is based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy, coupled with Raman spectroscopy, to qualify vesicular targets trapped on a microarray biochip.

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter ...

Fluorescence-Based Sorting of Adipose-Derived Extracellular Vesicles

Setting a Successful Sorting for Extracellular Vesicle Isolation

Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) contain a set of microRNAs with regenerative and anti-inflammatory roles. Therefore, purified MSC-EVs are envisioned as a next-generation therapeutic option for a wide array of diseases. In this protocol, we report the strategy for successfully sorting EVs from the supernatant of adipose-derived MSCs (ASCs), often used in orthopedics regenerative medicine applications.
First, we described the sample preparation, focusing on EV isolation and labeling steps with carboxyfluorescein succinimidyl ester (CFSE) for fluorescence detection; subsequently, we detailed the sorting process, which constitutes the main part of the protocol. 
In addition to the rules defined by MISEV 2023 and MIFlowCyt EV guidelines, we applied specific experimental conditions concerning nozzle size, frequency, and sheath pressure. Morphological parameters are established using beads of diameters selected to cover the theoretical range of EV size. After ASC-EVs sorting, we performed a purity check of the sorted fraction by re-analyzing it with the sorter and verifying the EV size distribution with the nanoparticle tracking analysis technique.
Due to the increasing importance of EVs, having a pure population to study and characterize is becoming crucial. Here, we demonstrate a winner strategy to set up sorting to achieve this goal.

This protocol provides a detailed description of sorting extracellular vesicles (EVs) released by mesenchymal stromal cells. In particular, it focuses on the instrument setting and the optimization of the sorting conditions. The goal is to sort extracellular vesicles while preserving their characteristics.

Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) contain a set of microRNAs with regenerative and anti-inflammatory roles. ...

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

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