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 C...

<ol>
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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exodisc+for+Rapid,+Size-Selective,+and+Efficient+Isolation+and+Analysis+of+Nanoscale+Extracellular+Vesicles+from+Biological+Samples.">Exodisc for Rapid, Size-Selective, and Efficient Isolation and Analysis of Nanoscale Extracellular Vesicles from Biological Samples.</a> ACS Nano. 11 (2), 1360-1370 (2017).</li><li>Kim, T. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FAST:+Size-Selective,+Clog-Free+Isolation+of+Rare+Cancer+Cells+from+Whole+Blood+at+a+Liquid-Liquid+Interface.">FAST: Size-Selective, Clog-Free Isolation of Rare Cancer Cells from Whole Blood at a Liquid-Liquid Interface.</a> Analytical Chemistry. 89 (2), 1155-1162 (2017).</li><li>Joshi, B. S., De Beer, M. A., Giepmans, B. N. G., Zuhorn, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis+of+Extracellular+Vesicles+and+Release+of+Their+Cargo+from+Endosomes.">Endocytosis of Extracellular Vesicles and Release of Their Cargo from Endosomes.</a> ACS Nano. 14 (4), 4444-4455 (2020).</li><li>Sung, B. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+live+cell+reporter+of+exosome+secretion+and+uptake+reveals+pathfinding+behavior+of+migrating+cells.">A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells.</a> Nature Communications. 11, 2092 (2020).</li><li>Heusermann, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+surf+on+filopodia+to+enter+cells+at+endocytic+hot+spots,+traffic+within+endosomes,+and+are+targeted+to+the+ER.">Exosomes surf on filopodia to enter cells at endocytic hot spots, traffic within endosomes, and are targeted to the ER.</a> Journal of Cell Biology. 213 (2), 173-184 (2016).</li><li>Reclusa, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+extracellular+vesicles+visualization:+From+static+to+motion.">Improving extracellular vesicles visualization: From static to motion.</a> Scientific Reports. 10, 6494 (2020).</li><li>Verweij, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Tracking+of+Inter-organ+Communication+by+Endogenous+Exosomes+In+Vivo.">Live Tracking of Inter-organ Communication by Endogenous Exosomes In Vivo.</a> Developmental Cell. 48 (4), 573-589 (2019).</li><li>Lai, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+and+tracking+of+tumour+extracellular+vesicle+delivery+and+RNA+translation+using+multiplexed+reporters.">Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters.</a> Nature Communications. 6, 7029 (2015).</li><li>Durak-Kozica, M., Baster, Z., Kubat, K., St&#281;pie&#324;, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+visualization+of+extracellular+vesicle+uptake+by+endothelial+cells.">3D visualization of extracellular vesicle uptake by endothelial cells.</a> Cellular &amp; Molecular Biology Letters. 23, 57 (2018).</li><li>Sunkara, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+Automated,+Label-Free+Isolation+of+Extracellular+Vesicles+from+Whole+Blood+for+Cancer+Diagnosis+and+Monitoring.">Fully Automated, Label-Free Isolation of Extracellular Vesicles from Whole Blood for Cancer Diagnosis and Monitoring.</a> Theranostics. 9 (7), 1851-1863 (2019).</li><li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internalization+of+trophoblastic+small+extracellular+vesicles+and+detection+of+their+miRNA+cargo+in+P-bodies.">Internalization of trophoblastic small extracellular vesicles and detection of their miRNA cargo in P-bodies.</a> Journal of Extracellular Vesicles. 9 (1), 1812261 (2020).</li><li>Dong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+evaluation+of+methods+for+small+extracellular+vesicles+separation+from+human+plasma,+urine+and+cell+culture+medium.">Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium.</a> Journal of Extracellular Vesicles. 10, 12044 (2020).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#38;L preadsorbed</td>
			<td>abcam</td>
			<td>&#160;ab7063</td>
			<td>Mouse IgG antibody for negatvie control of EV labeling</td>
		</tr>
		<tr>
			<td>Ibidi USA&#160;U DISH &#956;-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+ CO2/O2/N2 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...

Watch this Scientific Journal Video about Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis at JoVE.com

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 ...

This protocol provides a robust methodology for analyzing EVs at a cellular level, and a practical approach for efficient EV uptake and EV tracking analysis. This method of EV labeling eliminates co-precipitation of EVs and dye, thus enhancing the EV signals. EV uptake is measured by volumetric analysis to distinguish the internalized EVs from the superficial EVs.EVs are active cargo, thus, their uptake allows the transfer of biochemical molecules. For research on EV-based drug delivery and cancer therapy, this technique can be an assessment tool. EV uptake plays a role in intercellular communication and is investigated in various research fields, such as cancer biology, neuroscience, and drug delivery.This protocol potentially facilitates efficient EV uptake assay. For isolation of extracellular vesicles, or EVs, inject one milliliter of pre-processed cell culture media into the sample chamber of the nanofiltration-based microfluidic device. To operate the device, spin the device in the benchtop spinning machine at 3000 RPM for 10 minutes.After spinning, remove the fluid from the waste chamber by pipetting or aspirating, then repeat this procedure twice for processing an additional two milliliters of the cell culture media. Next, to wash the isolated EVs, inject one milliliter of PBS into the sample chamber and spin the device in the benchtop spinning machine. For immunofluorescent labeling of the EVs, inject one microgram per milliliter of the EV-specific antibody into the elution hole of the device containing 100 microliters of isolated EVs.Then incubate for one hour in the dark on a plate shaker to ensure the even distribution of the antibody across the sample. Next, attach an adhesive tape to the elution hole. Inject one milliliter of PBS into the sample chamber to wash out residual antibodies, then spin the device until the sample chamber is empty.Repeat the wash by injecting another milliliter of PBS into the sample chamber, and spinning the device as demonstrated previously. After removing the residual fluid from the chamber, pipette the fluorescently-labeled EVs from the membrane chamber into an amber tube or micro tube. Protect the tube from light until use.Seed four times 10 to the fourth PC3 cells into a 35-milliliter dish with one milliliter media. Allow the cells to adhere overnight in optimal cell culture conditions. The following day, wash the adhered cells twice with exosome-depleted media.After diluting the fluorescently-labeled EVs to the appropriate concentration using exosome-depleted media, add the diluted EVs to the adhered target cells and incubate for the desired experimental time. After removing non-internalized EVs by washing the cells thrice with exosome-free media, label the cytoplasm of the adhered cells with one microgram per milliliter of CMTMR, and incubate in optimal cell culture conditions. For live cell imaging, place the prepared cells in the onstage incubator.After setting the imaging parameters based on control samples, determine the depth of the target cells and the range of stacking size in the Z direction to acquire 3D confocal images, then set the image acquisition to multiple Z-stacked images of both cell-specific dye and EV-specific dye simultaneously. For image processing, utilize automatic image processing software. To build virtual surfaces of the cells, click on Add new Surfaces.To configure the virtual cell surfaces, click the Add button and select Quality as the filter type. Threshold the appropriate value for the low limit by visual inspection, set the maximum value for the upper limit, and click the Finish button Next, to build the virtual dots of the EVs, click the button Add new Spots. Under Algorithm Settings, select Different Spot Sizes and Shortest Distance Calculation, then click Next and select Channel 1 Alexa Fluor 488 as the source channel.Enter the appropriate value for the estimated XY diameter under Spot Detection and click Next. To configure the virtual EV dots, click the Add button and select Quality as the filter type. Set the lower threshold by a visual inspection and click Next For the Spot Regions Type, select Absolute Intensity, then click Next.To threshold the region of EV dots, enter the appropriate value for Region Threshold by visual inspection. Select Diameter From under Region Volume and click Finish. To split the grouped spots inside the built surface, click on the Build Spots and go into Filters.Next click the Add button and select Shortest Distance to Surfaces, Surfaces Surface 1 as the filter type. After setting the lowest threshold for the low limit and the appropriate value for the upper limit, click the Duplicate Section to the new Spots button. For automatic counting of EVs inside the cells, click the Built Spots 1 selection, go to Statistics, and export the value from the Total Number of Spots.To obtain the number of cells, click the Built Surfaces 1, then go to Statistics and export the value of Total Number of Surfaces from Overall. Finally, go to the Detailed tab under Statistics to export the volume. EVs isolated from PC3 cell culture media using a nanofiltration-based microfluidic device were labeled with a fluoro-4-conjugated EV-specific antibody, and visualized using 3D confocal microscopy.The internalized EVs were visualized as puncta and an individual EV.Post-processing of these images allows the visualization and quantification of EV internalization into the cells. The EV uptake assay results indicate that the level of EV uptake depends on the length of the incubation period. The number of internalized EVs can be normalized to the recipient cell volume to determine the actual rate of EV uptake for the specified cell.This procedure allows for the systematic exclusion of non-internalized EVs to precisely measure the number of internalized EVs. The size distribution of internalized EV dots is shown here. So the key steps are the utilization of the microfluidic device for EV labeling, the minimization of background fluorescence when visualizing fluorescent EVs, and then the post-imaging analysis to determine internalized versus superficial EVs.So intracellular EV tracking can be performed in real time on a 3D plane, and additionally, EV trafficking analysis on spatial/temporal resolutions and the co-localization of EVs with subcellular organelles can be achieved. So we believe this technique provides an easier, more efficient way to investigate EVs within the intra-and extracellular matrices for researchers within the cellular communication field for EVs.

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Endocytosis and Exocytosis

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6.8K 조회수.  Johns Hopkins University School of Medicine. 이 프로토콜은 셀룰러 수준에서 전기를 분석하기 위한 강력한 방법론과 효율적인 EV 섭취 및 EV 추적 분석을 위한 실용적인 접근 방식을 제공합니다. 이 EV 라벨링 방법은 EV와 염료의 공동 침전을 제거하여 EV 신호를 향상시킵니다. EV 섭취량은 피상적 인 전기 EV와 내부화된 전기를 구별하기 위해 체적 분석에 의해 측정됩니다.전기자동차는 활성 화물이므로, 이들의 섭취?...