Name: extracellular vesicle uptake assay via confocal microscope imaging analysis
Uploaded: 2022-02-14T13:00:00
Description: Watch this Scientific Journal Video about Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis at JoVE.com

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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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology,+function,+and+biomedical+applications+of+exosomes.">The biology, function, and biomedical applications of exosomes.</a> Science. 367 (6478), (2020).</li><li>Mathieu, M., Martin-Jaular, L., Lavieu, G., Th&#233;ry, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificities+of+secretion+and+uptake+of+exosomes+and+other+extracellular+vesicles+for+cell-to-cell+communication.">Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication.</a> Nature Cell Biology. 21 (1), 9-17 (2019).</li><li>Bonsergent, E., 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=Quantitative+characterization+of+extracellular+vesicle+uptake+and+content+delivery+within+mammalian+cells.">Quantitative characterization of extracellular vesicle uptake and content delivery within mammalian cells.</a> Nature Communications. 12, 1864 (2021).</li><li>Mulcahy, L. A., Pink, R. C., Carter, D. R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Routes+and+mechanisms+of+extracellular+vesicle+uptake.">Routes and mechanisms of extracellular vesicle uptake.</a> Journal of Extracellular Vesicles. 3 (1), 24641 (2014).</li><li>Dehghani, M., Gaborski, T. 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A., 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=Characterization+of+Uptake+and+Internalization+of+Exosomes+by+Bladder+Cancer+Cells.">Characterization of Uptake and Internalization of Exosomes by Bladder Cancer Cells.</a> BioMed Research International. 2014, 619829 (2014).</li><li>Feng, D., 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=Cellular+Internalization+of+Exosomes+Occurs+Through+Phagocytosis.">Cellular Internalization of Exosomes Occurs Through Phagocytosis.</a> Traffic. 11 (5), 675-687 (2010).</li><li>Hoen, E. N. M. N. -. 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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. 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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 ...

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