We would like to thank the NIH for the funding (EY031316 and EY034714). We would also like to thank UNTHSC and NTERI for the lab space.

All studies described adhered to the Declaration of Helsinki. Written consent was obtained from each subject prior to being included in the study. Institutional Review Board (IRB) approval from Aarhus University Hospital (1-10-72-77-14) and Dean McGee Institute (1576837-2) was received according to the federal and institutional guidelines. Before processing, all tear and saliva samples were de&#8211;identified. All studies were reviewed and approved by the North Texas Regional Institutional Review Board (#2020-030). The following protocol adheres to all guidelines and has been approved, as mentioned above.
1. Day 1: Sample preparation and incubation
<ol>
	<li>Collect biological fluid sample(s), i.e., tears and saliva from a subject(s) or thaw sample(s) from the freezer.
	<ol>
		<li>Tears: Collect tears passively from the lateral meniscus using a glass capillary tube.
		<ol>
			<li>Place the glass capillary tube on the lower eyelid, being careful not to touch the cornea.</li>
			<li>Collect a volume of 10 &#956;L of tears within 10 min.</li>
			<li>Use the tears immediately or store them at -20 &#176;C.</li>
		</ol>
		</li>
		<li>Saliva: Collect saliva from passive drool in a 1.5 mL microcentrifuge tube
		<ol>
			<li>Allow the donor to pool saliva in their mouth.</li>
			<li>Ask the donor to drool into the tube with a funnel.</li>
			<li>Use saliva immediately or store at -20 &#176;C.&#160;</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Place the tetraspanin chip(s) in the chip washer plate for 15 min before adding samples to allow them to reach room temperature. While placing the chips in the well, ensure the numbers are on the bottom side facing up and that the chip(s) do not touch the side walls of the well(s).</li>
	<li>Record the ID number and chip number in the notebook to keep track of the chip(s).</li>
	<li>Plug in the universal serial bus (USB) that comes with the kit, and extract and save the folder with the associated kit number to a designated area of your choosing.</li>
	<li>Pipette 99 &#956;L of Solution B in a 1.5 mL tube.</li>
	<li>Add 1 &#956;L of biological fluid into the 1.5 mL tube prepared in the previous step.</li>
	<li>Spin down the tubes.</li>
	<li>Take 70 &#956;L of the solution from step 1.6 and pipette on the tetraspanin chip.</li>
	<li>Place the film on the chip washing plate to prevent the sample from evaporating.</li>
	<li>Incubate for 16 h at room temperature (RT) on a vibration&#8211;free bench.</li>
</ol>
2. Day 2: Chip washing
<ol>
	<li>Turn on the chip washing machine.</li>
	<li>Remove the film from step 1.9 from the plate.</li>
	<li>Place the plate in the chip washer, making sure it locks into place, and close the lid. 
	NOTE: The plate will not move easily if correctly locked into place.</li>
	<li>Press CW-TETRA v0 option on the chip washer.</li>
	<li>Select the starting row and the number of rows to wash.</li>
	<li>Press Continue on the chip washing machine.</li>
	<li>While the chip(s) are being washed, prepare the blocking solution containing the antibodies in a tube as follows:
	<ol>
		<li>Prepare 300 &#181;L of Blocking solution per chip.</li>
		<li>Add 0.6 &#181;L or each antibody per chip (vortex and spin down antibodies before adding to the blocking solution)</li>
		<li>Vortex the blocking solution + antibody cocktail. 
		NOTE: Cover the blocking solution + antibody cocktail from light to ensure antibody stability. This must be made on the day of use.</li>
	</ol>
	</li>
	<li>When the chip washer chirps, add 250 &#181;L of the blocking solution + antibody cocktail on top of the chip(s).</li>
	<li>Close the lid and press Continue. 
	NOTE: The chip washer will now do a 1&#8211;h incubation along with several additional rounds of washing and rinsing.</li>
	<li>When the chip washer chirps, remove the chip washing plate and move the chip(s) up the ramp.</li>
	<li>Place the plate back into the chip washer, making sure it locks into place. Close the lid and press Continue.</li>
	<li>When the chip washer completes the program, remove the plate and place the chip(s) onto a paper towel. Cover the chip(s) from the light.</li>
</ol>
3. Day 2: Scanning the chip(s)
<ol>
	<li>Turn on the scanner and open the scanning software. 
	NOTE: The scanner will undergo self&#8211;checks to indicate if there are any errors or if scanning can be continued.</li>
	<li>Select Save Folder and choose the location to save the files.</li>
	<li>Select ChipFile Folder and choose the folder saved in step 1.4.</li>
	<li>Select the drop-down option to choose each chip and locate it in Chip Position on the chuck. Repeat this step for all the chips that need to be scanned.</li>
	<li>Turn on the fluorescence by clicking the three squares below Chip Position. Squares will turn yellow, blue, and red, respectively.</li>
	<li>Remove the chuck lid by pressing the lid down and pull it.</li>
	<li>Place the chip(s) on the chuck, ensuring they are firmly secured.</li>
	<li>Place the chuck lid back on by placing the pegs in the holes, press down, and slide the lid to lock it into place.</li>
	<li>Place the chuck onto the stage, making sure it locks into place.</li>
	<li>Select Scan Chips.</li>
	<li>Select OK.</li>
	<li>Once the stage has fully moved into the machine, close the door. 
	NOTE: It takes approximately 12 min to scan a single chip.</li>
	<li>Once the program is done scanning the chip(s), open the door to eject the stage check to see if the scans were successful.
	<ol>
		<li>If the scans were successful, discard the chip(s) and place the next set of chips on the chuck, or return the empty chuck to the stage. Then, exit the software and turn off the machine when done.</li>
		<li>If the scans were not successful, look at the error code and correct the error.</li>
	</ol>
	</li>
</ol>
4. Data processing
<ol>
	<li>Open the analyzing software.</li>
	<li>Select Prescan Data and choose the folder that has the scan from Day 2 step 3.2.</li>
	<li>Select Next.</li>
	<li>Label the chip(s) in the Sample Name column with the name of the sample by clicking the cell and typing in the sample ID.</li>
	<li>Next, label the tetraspanins in the Channel Name column. Red = CD63, Green = CD81, and Blue = CD9.</li>
	<li>Select Next.</li>
	<li>Select High CV from the Select Spot group to inspect the drop&#8211;down box and turn off the high CVs for each chip by disabling one or two spots.</li>
	<li>Next, select High Count and try to turn off the high&#8211;count warnings. 
	NOTE: Some samples may have too high of a count, but they are still able to be analyzed.</li>
	<li>Select All Spots from the drop-down menu, select Spot Montage, and visually check each chip for any deformities or scratches that may be present.</li>
	<li>Select Next.</li>
	<li>In the CD63 channel, set the minimum to 300 to get rid of red cells in the Avg % Included row; yellow cells are fine, and proceed to the CD81 channel. If setting the minimum value to 300 does not change the red cell to yellow or white, proceed to step 4.14 for troubleshooting.</li>
	<li>In the CD81 channel, set the minimum to 300 to get rid of red cells in the Avg % Included row; yellow cells are fine, and proceed to the CD9 channel. If setting the minimum value to 300 does not change the red cell to yellow or white, proceed to step 4.14 for troubleshooting.</li>
	<li>In the CD9 channel, set the minimum to 400 to get rid of red cells in the Avg % Included row; yellow cells are fine, and proceed to step 4.15. If setting the minimum value to 400 does not change the red cell to yellow or white, proceed to step 4.14 for troubleshooting.</li>
	<li>Troubleshooting
	<ol>
		<li>Increase the minimum value by 100 units.</li>
		<li>If step 4.14.1 did not work, then select the chip in the Pick Chips to work on section and choose the chip. Select Particle Count and disable the spot(s) that has high particle counts. Select Intensity/Size, select All Chips, and see if this resolves the issue.</li>
		<li>If step 4.14.2 did not work, select the chip in the Pick Chips to work on section and choose the chip. Select Single-Chip Cutoffs and increase the minimum by 100 units. Then select All Chips to see if the red cell is now yellow. If this does not work, repeat this step until the Avg % Included cell is now yellow or white.</li>
	</ol>
	</li>
	<li>Select Next.</li>
	<li>Deselect the IM channel.</li>
	<li>Set the heatmap value in the bottom right-hand corner to include all particles and select Add Plot to Report.
	<ol>
		<li>Adjust the maximum number in the drop-down box and manually enter a value.</li>
	</ol>
	</li>
	<li>Next, select the drop-down menu in the top middle under Capture Probes and select CD63.</li>
	<li>Select Add Plot to Report to add the total particle count to the report.</li>
	<li>Select the drop&#8211;down menu under Analysis Mode, choose Colocalization, and Add Plot to Report.</li>
	<li>Next, select Size, select Change view to Plot, and select Add Plot to Report.</li>
	<li>Finally, select Images and select Add Plot to Report.</li>
	<li>Repeat steps 4.18&#8211;4.22 for CD81 and CD9, making sure to deselect the IM channel each time.</li>
	<li>Once all items have been added to the report, select Export Report, make sure it saves to the correct file, and select Folder. 
	NOTE: Excel files and images have been generated and can now be used for further analysis.</li>
</ol>

The authors have no competing financial interests or other conflicts of interest to disclose.

The most critical step in this protocol is to make sure that an optimal concentration of EVs is achieved. There needs to be enough EVs present to obtain a reading, but not too many EVs that will oversaturate the chip. The best way to determine the optimal EV concentration is to do an optimization run with 1 &#181;L of sample and see if the concentration needs to be adjusted. Another critical step is to see if there is an abundance of cell debris in the sample, which can be determined by viewing large chunks on the chips in the analyzing software. If the sample has cell debris, a simple centrifugation or filtering of the sample should resolve this issue.
An additional, crucial step is ensuring that the chip(s) do not touch the walls of the well and avoiding contact with the center area of the chip(s) when placing them up the ramp. The scanner will read from the square at the center of the chip, so it is essential to avoid touching that area with the forceps to prevent disrupting the EVs.
This method does have some limitations such as having insufficient concentration of EVs for detection by the instrument. The concentration could be improved by either drying the sample or using a concentrator tube. Another limitation is that the analyzing software only measures EVs in the range of 50-200 nm, excluding some microvesicles and all apoptotic bodies from the size measurements75.
There are many ways to isolate (Table 1&#160;42,43,44,45,46,47,48,49,50,51,52, 53,54,55,56,57,58,59,60, 61,62,63,64,65,66,67) and analyze EVs (Table 2&#160;78,79,80,81,82,83,84,85,86,87,88,89,90), and the current gold standard for EV isolation is ultracentrifugation36,37,38to pellet the cells for assays such as Western Blots68,69,70and PCR71,72,73,74,75,76. While this protocol works well for large samples42,44,46, obtaining biological fluids can be challenging, and often, only a small volume of samples can be gathered at a time, which is not ideal for ultracentrifugation. In contrast, using this nanoparticle analyzer allows the user to generate valuable data, such as size, total particle count, and phenotype of the EVs, in as little as 1 &#181;L of sample, making it ideal for biological fluids. We will be able to expand the knowledge of EVs from small volumes of samples that are difficult to extract, which can increase patient comfort and possibly find potential therapeutic targets for various diseases and disorders.

Cells communicate with neighboring cells through various signaling mechanisms, including the release of extracellular vesicles (EVs), which play a key role in intercellular communication. EVs participate in intercellular communication by passing genetic cargo, such as DNA, RNA, and proteins, from one cell to another1,2,3,4,5. Currently, there are three categories of EVs: exosomes, microvesicles, and apoptotic bodies, which are characterized by their size. Exosomes are the smallest, with a diameter of 30-150 nm6,7,8, and are formed from the endosomal membrane system9,10,11 (Figure 1). Microvesicles are larger than exosomes in that they range from 100-1000 nm12,13,14 and buds off the plasma membrane11,12,13 (Figure 1). Apoptotic bodies are the largest of the EVs and range from 1000-5000 nm12,14,15, and they also bud off the plasma membrane12,16,17 (Figure 1). Apart from the size, EVs can be classified based on other biophysical characteristics, which include density, molecular markers such as CD63, CD81, CD9, and also biogenesis mechanism18. EVs can travel short distances between adjacent cells and long distances throughout the body19,20,21,22,23. EVs can be found in biological fluids such as blood24,25,26, cerebral spinal fluid27,28,29, tears30,31,32, and saliva33,34,35, to name a few.
To date, ultracentrifugation is one of the best-known methods for isolating EVs from samples36,37,38. This method requires multiple rounds of centrifugations and ultracentrifugation which can be carried out by increasing the speed to isolate the EVs from cells and cell debris. This method can be started with the low speed with pellet cells that will be followed by medium speed to eliminate larger vesicles and, finally an ultracentrifugation step to pellet EVs18. While ultracentrifugation is considered the best method for isolation, some limitations still exist in that it changes the morphology of the EVs39,40,41. Another technique used to isolate EVs is flow cytometry, which highlights the advantages of multiple time point and endpoint evaluation and high throughput single EV analysis. However, the limitations of flow cytometry include but are not limited to, clogging of the pores and weak signals. Another approach used is gradient centrifugation, which uses materials with different densities to be centrifuged with the EVs and allows better separation of the EVs compared to ultracentrifugation. Although this technique improves separation, it is labor intensive, time-consuming, and can lead to significant loss of sample. Additionally, precipitation and filtration can also be used to isolate EVs. Both these techniques are simple and fast, but both can lead to sample contamination. While there are several techniques to isolate EVs, each technique has its advantages and limitations, which are listed below (Table 1)&#160;42,43,44,45,46,47,48,49,50,51,52,53,54,55, 56,57,58,59,60,61,62,63,64,65,66,67.
Once the EVs have been isolated, molecular assays can be performed to characterize the EVs. Western blots are a common assay to look for surface and cargo protein expression68,69,70 and polymerase chain reaction (PCR) is used for miRNA expression71,72,73 for EVs74,75,76. These assays are established and can generate intriguing results. A limitation of these methods is that they require a large quantity of proteins or RNA from EVs to get a reading77, which is a problem for samples that have a small volume or EV concentration, to begin with.
The nanoparticle analyzer discussed in this paper allows the user to overcome many of the limitations stated in Table 1 and Table 2&#160;78,79,80,81,82,83,84,85,86,87,88,89,90. This method does not require the utilization of isolation techniques, which will help overcome the reduced yield of EVs. This method also allows the user to analyze the surface and cargo proteins, total EV count, and EV size from a sample volume of as little as 1 &#181;L. This is done by using tetraspanin chips provided by the company that uses an antibody microarray with tetraspanin antibodies, CD63, CD81, and CD9, to identify EVs in a solution, as shown in Figure 2. Fluorescent antibodies confirm the presence of EVs as well as preventing contaminating particles from skewing the results.
The overall goal of this technique is to provide a less time-consuming method to analyze EVs as well as analyze EVs from a small volume of sample. Using this nanoparticle analyzer allows the users to analyze the size, total particle count, and surface proteins from as little as 1 &#181;L of a sample, which is ideal for biological fluids such as tears and saliva.

<table><tbody><tr><td>ChipWasher 100</td><td>NanoView</td><td>EV-CW100</td><td>Incubates, washes, rinses and dries the tetraspanin chips. This current model is no longer available. Price at time of purchase: $9,995.00&amp;nbsp;</td></tr><tr><td>ExoView Analyzer software</td><td>NanoView</td><td>N/A</td><td>Analyzes the chip informations and produces excel files for further analysis. No longer available.</td></tr><tr><td>ExoView R100</td><td>NanoView</td><td>EV-R100</td><td>Used to scan the tetraspanin chips at 3 wavelengths. This current model is no longer available. Price at time of purchase:&amp;nbsp; $110,000.00</td></tr><tr><td>ExoView Scanner software</td><td>NanoView</td><td>N/A</td><td>Scans the chips at 3 different wavelengths. No longer available.</td></tr><tr><td>Human Tetraspanin Kits</td><td>Unchained Labs</td><td>EV-TETRA-C</td><td>Includes 8 tetraspanin chips, Incubation Solution, Blocking Solution, CD63 antibody, CD81 antibody, CD9 antibody, Solution A, Solution B, USB, and plate cover.</td></tr></tbody></table>

characterizing extracellular vesicles from biological fluids

Extracellular vesicles (EVs) are structures that are produced from cells and participate in intercellular communication by transporting biomolecules from one cell to another. EVs have been shown to travel short and far distances in the body and are tissue-specific. EVs are not only found in tissues, but they can also be found in practically all bodily fluids, such as tears, saliva, cerebral spinal fluid, blood, etc. Even though EVs can be collected non-invasively from tears and saliva, only small volumes can be collected at a time, which can cause issues in obtaining enough EVs to analyze proteins. The scanner discussed in this paper is a nanoparticle analyzer that provides a solution to&#160;this problem, allowing us to characterize and study the phenotype, size, and total particle count of EVs from as little as 1 &#181;L of biological fluid. This protocol will expand the knowledge of EVs from small volumes of samples that are difficult to extract from patients. This could enhance patient comfort and potentially identify new therapeutic targets for a range of diseases and disorders.

When analyzing the spots on the chips, aim for an optimal concentration of EVs by observing fluorescently labeled EVs in the CD63, CD81, and CD9 channels, and the MIgG channel30,91 should remain black, as this is the control channel as seen in Figure 3A. CD81 will be reduced for biological fluids. If there is no fluorescence in the CD63, CD81, and CD9 channels, there is an absence of EVs (Figure 3B). Be careful not to oversaturate the chip (Figure 3C). This will make it difficult for the analyzing software to calculate the accuracy of the size, total particle count, and phenotypes of the EVs. From the spots of the optimal concentration, the analyzing software will be able to measure the size (50-200 nm), total particle count, and tetraspanin expression of the EVs (Figure 4) and will be exported into individual spreadsheets for further analysis. The tetraspanin analysis will include the colocalization of the surface proteins on the EVs. The colocalization will be represented with a &#34;/&#34;, for example, &#34;CD63/CD81&#34;. This does not mean that CD63 and CD81 are combined; it means that both CD63 and CD81 are located on the surface of the EV.
These results will provide valuable insight between healthy and diseased samples. We will be able to determine if healthy samples or diseased samples produce more EVs, larger or smaller EVs, and the phenotype of the EVs. Any or all of these characteristics could play a role in the disease biogenesis and/or progression. With these results, we will be able to see if there is an absence or increase of the tetraspanin levels, which can provide insight into cell processes and the formation of EVs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67699/67699fig1.jpg" /> 
Figure 1: The types and sizes of the different EVs. <a href="https://www.jove.com/files/ftp_upload/67699/67699fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67699/67699fig2.jpg" /> 
Figure 2: Schematic of how the tetraspanin chips capture and detect EVs. <a href="https://www.jove.com/files/ftp_upload/67699/67699fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67699/67699fig3.jpg" /> 
Figure 3: Optimizing EV concentrations. (A) Optimal concentration of EVs. (B) Negative result: no EVs present. (C) Oversaturation of EVs. <a href="https://www.jove.com/files/ftp_upload/67699/67699fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67699/67699fig4.jpg" /> 
Figure 4: Data generated by the nanoparticle analyzer. (A) EV diameter size measured in nanometers. (B) Total particle count of EVs. (C) EV colocalized phenotype. <a href="https://www.jove.com/files/ftp_upload/67699/67699fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Technique</td>
			<td>Advantages</td>
			<td>Limitations</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>High Purity42,43</td>
			<td>Changes EV morphology42,44</td>
		</tr>
		<tr>
			<td></td>
			<td>Homogeneity42,45</td>
			<td>Requires large volume42,44,46</td>
		</tr>
		<tr>
			<td></td>
			<td>Functionality42,43</td>
			<td>Expensive42,43</td>
		</tr>
		<tr>
			<td>Flow Cytometry</td>
			<td>Evaluate samples at multiple time points47</td>
			<td>Weak signal48,49</td>
		</tr>
		<tr>
			<td></td>
			<td>Multiple endpoints47,50</td>
			<td>Clogged pores51</td>
		</tr>
		<tr>
			<td></td>
			<td>High throughput single EV analysis52,53</td>
			<td></td>
		</tr>
		<tr>
			<td>Density Gradient Centrifugation</td>
			<td>Produce highly pure samples54</td>
			<td>Labor intensive55,56</td>
		</tr>
		<tr>
			<td></td>
			<td>Variety of samples55,57</td>
			<td>Time-consuming55,58&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Significant yield loss54</td>
		</tr>
		<tr>
			<td>Precipitation</td>
			<td>Simple59,60</td>
			<td>Contamination36,44,61</td>
		</tr>
		<tr>
			<td></td>
			<td>Fast61,62</td>
			<td>Non-Specific63</td>
		</tr>
		<tr>
			<td></td>
			<td>High Yield44,59,62</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Neutral pH36</td>
			<td></td>
		</tr>
		<tr>
			<td>Filtration</td>
			<td>Simple64&#160;</td>
			<td>Trap exosomes65</td>
		</tr>
		<tr>
			<td></td>
			<td>Fast64</td>
			<td>Damages large vesicles36</td>
		</tr>
		<tr>
			<td></td>
			<td>Inexpensive64</td>
			<td>Filter cake66,67</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Exosome clogging65</td>
		</tr>
	</tbody>
</table>
Table 1: Advantages and Limitations of EV isolation techniques. A list of the various ways to isolate EVs, including their advantages and limitations.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Technique</td>
			<td>Advantages</td>
			<td>Limitations</td>
		</tr>
		<tr>
			<td>Dynamic light scattering</td>
			<td>Measure particles ranging in size 1 nm to 6 &#181;M78&#160;</td>
			<td>Not suitable for measuring complex exosome samples with large size range79&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td>The lower limit is 10 nm suitable for monodisperse systems79&#160;</td>
			<td>Not able to distinguish contaminated proteins from exosomes79</td>
		</tr>
		<tr>
			<td>Transmission electron microscopy (TEM)</td>
			<td>Observe morphology of exosomes79,80&#160;</td>
			<td>Complicated sample preparations79&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td>Observe internal structure81&#160;</td>
			<td>Not able to distinguish exosomes based on size and shape because of exaggerated fluorescence signals82&#160;</td>
		</tr>
		<tr>
			<td>Nanoparticle Tracking analysis</td>
			<td>Measure concentration, size and size distribution of exosomes in the 10 nm to 2 &#181;M range78&#160;</td>
			<td>Cannot differentiate the EVs from protein aggregates and other contaminants83&#160;</td>
		</tr>
		<tr>
			<td rowspan="2"></td>
			<td>Quick sample preparation and measurement78,84</td>
			<td>Expensive NTA instrument85&#160;</td>
		</tr>
		<tr>
			<td>Samples can be recovered in native form84&#160;</td>
			<td>Sensitive to vibrations85</td>
		</tr>
		<tr>
			<td>Western Blot (WBs)</td>
			<td>can qualitatively and quantitatively analyze marker proteins79,86&#160;</td>
			<td>Complicated and time consuming79&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td>Analyzing exosomes from cell culture media79&#160;</td>
			<td>EV isolates may contain lipoproteins and other contaminants86&#160;</td>
		</tr>
		<tr>
			<td rowspan="2">Flow cytometry</td>
			<td>Higher sensitivity and high-resolution imaging to</td>
			<td rowspan="2">Time-consuming and laborious with detection limit of 400 nm79,88&#160;</td>
		</tr>
		<tr>
			<td>distinguish stained exosomes from containments87&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td>Required low sample concentration79&#160;</td>
			<td>Optical signals hinder the accuracy and resolution88&#160;</td>
		</tr>
		<tr>
			<td>Exoview</td>
			<td>Measure Tetraspanins (CD9, CD63, and CD81) on exosomes89&#160;</td>
			<td>Does not measure larger EV sizes75</td>
		</tr>
		<tr>
			<td></td>
			<td>Measures EV cargo proteins90</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Advantages and limitations of EV characterization techniques.

Here, we demonstrate the characterization method for extracellular vesicles (EVs) collected from biological fluids, such as tears and saliva, of human subjects. The scanner used in this method is capable of detecting the phenotype, size, and total particle count of EVs from 1 &#181;L of the sample.

Characterizing Extracellular Vesicles from Biological Fluids

Extracellular vesicles (EVs) are structures that are produced from cells and participate in intercellular communication by transporting biomolecules from ...

characterizing-extracellular-vesicles-from-biological-fluids

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JoVE Journal

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139 Views.  University of North Texas Health Science Center. Here, we demonstrate the characterization method for extracellular vesicles (EVs) collected from biological fluids, such as tears and saliva, of human subjects. The scanner used in this method is capable of detecting the phenotype, size, and total particle count of EVs from 1 µL of the sample.

Video: Characterizing Extracellular Vesicles from Biological Fluids

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma.

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving ...

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 ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...

Isolation and Characterization of Cyanobacterial Extracellular Vesicles

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential biotechnology models. Recent work has demonstrated that both marine and freshwater cyanobacteria produce extracellular vesicles -&#160;small membrane-bound structures released from the outer surface of the microbes. While vesicles likely contribute to diverse biological processes, their specific functional roles in cyanobacterial biology remain largely unknown. To encourage and advance research in this area, a detailed protocol is presented for isolating, concentrating, and purifying cyanobacterial extracellular vesicles. The current work discusses methodologies that have successfully isolated vesicles from large cultures of Prochlorococcus, Synechococcus, and Synechocystis. Methods for quantifying and characterizing vesicle samples from these strains are presented. Approaches for isolating vesicles from aquatic field samples are also described. Finally, typical challenges encountered with cyanobacterial vesicle purification, methodological considerations for different downstream applications, and the trade-offs between approaches are also discussed.

The present protocol providesdetailed descriptions for isolation, concentration,and characterization of extracellular vesicles from cyanobacterial cultures. Approaches for purifying vesicles from cultures at different scales, trade-offs among methodologies, and considerations for working with field samples are also discussed.

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential ...

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs&#39; size was 30-200 nm, with a particle concentration of 7.75 &#215; 1010 particles/mL and a protein concentration of 80 &#181;g/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells&#39; small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this ...

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

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.

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...

Characterizing Extracellular Vesicles from Biological Fluids

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Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Isolation and Characterization of Cyanobacterial Extracellular Vesicles

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

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