This work was supported by the National Institutes of Health (ES030973-01A1, R01ES025225, R01DK066525, P30DK026687, P30DK063608). We acknowledge Jeffrey Bodycomb, Ph.D. of HORIBA Instruments Incorporated for his help calibrating the instrument.

All work with these samples was performed in compliance with Institutional Animal Care and Use Committee and Institutional Review Board guidelines. A schematic overview of the NTA method is depicted in Figure 2.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62447/62447fig02.jpg" /> 
Figure 2: Overview of NTA method using the particle tracking instrument. The sample is...

<ol>
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Association of plasma membrane activities with released vesicles (exosomes).</a> The Journal of Biological Chemistry. 262, 9412-9420 (1987).</li><li>Th&#233;ry, C., Ostrowski, M., Segura, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+vesicles+as+conveyors+of+immune+responses.">Membrane vesicles as conveyors of immune responses.</a> Nature Reviews Immunology. 9, 581-593 (2009).</li><li>Y&#225;&#241;ez-M&#243;, M., 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=Biological+properties+of+extracellular+vesicles+and+their+physiological+functions.">Biological properties of extracellular vesicles and their physiological functions.</a> Journal of Extracellular Vesicles. 4, 27066 (2015).</li><li>Lo Cicero, A., Stahl, A., Raposo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+shuffling+intercellular+messages:+for+good+or+for+bad.">Extracellular vesicles shuffling intercellular messages: for good or for bad.</a> Current Opinion in Cell Biology. 35, 69-77 (2015).</li><li>Raposo, G., Stoorvogel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles:+exosomes,+microvesicles,+and+friends.">Extracellular vesicles: exosomes, microvesicles, and friends.</a> The Journal of Cell Biology. 200, 373-383 (2013).</li><li>Mathivanan, S., Ji, H., Simpson, R. 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All authors declared that there are no conflicts of interest.

Here, we demonstrate a protocol for NTA of EVs to measure the size distribution of a wide range of particle sizes simultaneously and measure total EV concentration in a polydisperse sample. In this study, mouse perigonadal adipose tissue and human plasma were used as the source of EVs. However, EVs isolated from other tissues or biological fluids such as serum, urine, saliva, breast milk, amniotic fluid, and cell culture supernatant may also be used for NTA. Measurements of polystyrene bead standards ensured that the ins...

Extracellular vesicles (EVs) are small (0.03-2 &#181;m) membrane-bound vesicles secreted by nearly all cell types1. They are often referred to as &#34;exosomes,&#34; &#34;microvesicles,&#34; or &#34;apoptotic bodies&#34; depending on their mechanism of release and size2. While it was initially thought that EVs were simply a means of eliminating waste from the cell to maintain homeostasis3, we now know that they can also participate in intercellular communication via transfer of molecular material - including DNA, RNA (mRNA, microRNA), lipids, and proteins4,5 - and that they are important regulators of normal physiology as well as pathological processes1,5,6,7,8.
There are many different methods to isolate and quantify EVs, which have been described elsewhere9,10,11,12. The isolation protocol used as well as the source of EVs can greatly impact EV yield and purity. Even differential centrifugation, long considered the &#34;gold standard&#34; approach for exosome isolation, can be subject to substantial variability subsequently impacting the EV population obtained and downstream analyses13. Thus, the various different methodologies for EV isolation and quantification make it difficult to compare, reproduce, and interpret results of experiments reported in the literature14.&#160;Furthermore, EV release can be regulated by cellular conditions or various external factors. It has been suggested that EVs play a role in maintaining cellular homeostasis by protecting cells against intracellular stress15, as several studies have shown that cellular stress stimulates EV secretion. For example, increased EV release has been reported after cellular exposure to hypoxia, endoplasmic reticulum stress, oxidative stress, mechanical stress, cigarette smoke extract, and particulate matter air pollution16,17,18,19,20,21,22. EV release has also been shown to be modified in vivo; mice subjected to a high fat diet or fasting for sixteen hours released more adipocyte EVs23. To investigate whether a specific treatment or condition alters EV release, the number of EVs must be accurately determined. Assessment of the EV size distribution may also indicate the predominant subcellular origin of EVs (e.g., fusion of late endosomes/multivesicular bodies with the plasma membrane vs. budding of the plasma membrane)24. Thus, there is a need for robust methods to accurately measure the total concentration and the size distribution of the EV prep being studied.
A rapid and highly sensitive method for the visualization and characterization of EVs in solution is nanoparticle tracking analysis (NTA). A detailed explanation of the principles of this method and comparison with alternative methods for assessment of EV size and concentration have been described previously25,26,27,28. Briefly, during NTA measurement, EVs are visualized by the light scattered when they are irradiated with a laser beam. The scattered light is focused by a microscope onto a camera which records the particle movement. The NTA software tracks the random thermal motion of each particle, known as Brownian motion, to determine the diffusion coefficient which is used to calculate the size of each particle using the Stokes-Einstein equation. NTA was first applied to measurement of EVs in a biological sample in 201125. Until recently, there were only two mainstream companies offering commercial NTA instruments29 until the introduction of the ViewSizer 3000 (hereafter referred to as the particle tracking instrument) which uses a combination of novel hardware and software solutions to overcome significant limitations of other NTA techniques.
The particle tracking instrument characterizes nanoparticles in liquid samples by analyzing their Brownian motion and characterizes larger micron-sized particles by analyzing gravitational settling. This instrument&#39;s unique optical system, which includes multispectral illumination with three laser light sources (at 450 nm, 520 nm, and 635 nm), allows researchers to analyze a wide range of particle sizes (e.g., exosomes, microvesicles) simultaneously. A schematic of the instrument setup is shown in Figure 1.
Here, we demonstrate how to perform particle size distribution and concentration measurements of isolated mouse and human EVs using a novel NTA instrument.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62447/62447fig01.jpg" /> 
Figure 1: Particle tracking instrument optical system. The NTA instrument illuminates particles using three lasers with the following wavelengths: 450 nm, 520 nm, 635 nm. Video recording of the scattered light from individual particles is detected and tracked by a digital video camera oriented 90&#176; from the cuvette. <a href="https://www.jove.com/files/ftp_upload/62447/62447fig01large.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>1X dPBS</td>
			<td>VWR</td>
			<td>02-0119-1000</td>
			<td>To dilute samples</td>
		</tr>
		<tr>
			<td>100 nm bead standard</td>
			<td>Thermo Scientific</td>
			<td>3100A</td>
			<td>To test ViewSizer 3000 calibration</td>
		</tr>
		<tr>
			<td>400 nm bead standard</td>
			<td>Thermo Scientific</td>
			<td>3400A</td>
			<td>To test ViewSizer 3000 calibration</td>
		</tr>
		<tr>
			<td>Centrifugal Filter Unit</td>
			<td>Amicon</td>
			<td>UFC901024</td>
			<td>To filter PBS diluent</td>
		</tr>
		<tr>
			<td>Collection tubes, 2 mL</td>
			<td>Qiagen</td>
			<td>19201</td>
			<td>For isolation of human plasma extracellular vesicles</td>
		</tr>
		<tr>
			<td>Compressed air duster</td>
			<td>DustOff</td>
			<td>DPSJB-12</td>
			<td>To clean cuvettes</td>
		</tr>
		<tr>
			<td>Cuvette insert</td>
			<td>HORIBA Scientific</td>
			<td>-</td>
			<td>Provided with purchase of ViewSizer 3000</td>
		</tr>
		<tr>
			<td>Cuvette jig</td>
			<td>HORIBA Scientific</td>
			<td>-</td>
			<td>To align magnetic stir bar while placing inserts inside cuvette; Provided with purchase of ViewSizer 3000</td>
		</tr>
		<tr>
			<td>De-ionized water</td>
			<td>VWR</td>
			<td>02-0201-1000</td>
			<td>To clean cuvettes</td>
		</tr>
		<tr>
			<td>Desktop computer with monitor, keyboard, mouse, and all necessary cables</td>
			<td>Dell</td>
			<td>-</td>
			<td>Provided with purchase of ViewSizer 3000</td>
		</tr>
		<tr>
			<td>Ethanol (70-100%)</td>
			<td>Millipore Sigma</td>
			<td>-</td>
			<td>To clean cuvettes</td>
		</tr>
		<tr>
			<td>ExoQuick ULTRA</td>
			<td>System Biosciences</td>
			<td>EQULTRA-20A-1</td>
			<td>For isolation of human plasma extracellular vesicles</td>
		</tr>
		<tr>
			<td>Glass scintillation vials with lids</td>
			<td>Thermo Scientific</td>
			<td>B780020</td>
			<td>To clean cuvettes</td>
		</tr>
		<tr>
			<td>&#34;Hook&#34; tool</td>
			<td>Excelta</td>
			<td>-</td>
			<td>Provided with purchase of ViewSizer 3000</td>
		</tr>
		<tr>
			<td>Lint-free microfiber cloth</td>
			<td>Texwipe</td>
			<td>TX629</td>
			<td>To clean cuvettes and cover work surface</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes, 2 mL</td>
			<td>Eppendorf</td>
			<td>22363344</td>
			<td>For isolation of human plasma extracellular vesicles</td>
		</tr>
		<tr>
			<td>Stir bar</td>
			<td>Sp Scienceware</td>
			<td>F37119-0005</td>
			<td></td>
		</tr>
		<tr>
			<td>Suprasil Quartz cuvette with cap</td>
			<td>Agilent Technologies</td>
			<td>AG1000-0544</td>
			<td>Initially provided with purchase of ViewSizer 3000</td>
		</tr>
		<tr>
			<td>ViewSizer 3000</td>
			<td>HORIBA Scientific</td>
			<td>-</td>
			<td>Nanoparticle tracking instrument</td>
		</tr>
	</tbody>
</table>

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.

Before this demonstration, the calibration of the instrument was first tested to ensure the validity of the acquired data by measuring the size distribution of polystyrene bead standards. We tested the size distribution of 100 nm and 400 nm beads using the default recording parameters and the processing settings recommended in this protocol (Figure 8).
For the 100 nm polystyrene bead standard, a concentration of 4.205 x 107 particles/mL was measured. Th...

Watch this Scientific Journal Video about Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles at JoVE.com

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

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 ...

NTA results are very prone to operator bias. This protocol demonstrates the effects of altered NTA parameters on obtained results. A standardized method will help increase rigor and reproducibility in NTA analysis.Analyzing the sample in a cuvette allows for a statistically random sample to be captured in each video. This results in more reproducible data and the visualization of particles over a wide range of sizes. As getting a sample in the recommended particle concentration range can be difficult, be sure to perform a serial dilution to identify the ideal dilution factor.Demonstrating the procedure will be Kungheng Cai, a PhD student from Anthony Ferrante's laboratory. To prepare cuvette for nanoparticle tracking analysis, cover the workspace with a lint-free material to prevent fibers from entering the cuvettes. Wearing gloves, place a cuvette containing a stir bar onto the magnetic cuvette jig.Use a hook tool to place the insert into the cuvette with the notch of the insert visible at the front of the cuvette. Use a pipette to slowly add 400 to 500 microliters of purified extracellular vesicles onto the cuvette through the hole in the insert and mix the sample by gently pipetting without introducing air bubbles, then cap the cuvette, tapping out bubbles as necessary and use a lint-free cloth to wipe the outside surface of the cuvette. To analyze the particle concentration of the diluent or a sample, turn on the computer workstation and instrument and start the particle tracking analysis program.When prompted, click NTA and open the recording tab. Follow the onscreen instructions to fill out all the necessary sample information. For EV tracking analysis, set the diluent to PBS.The salinity will auto-populate to 9%To obtain the diluent or sample particle concentration, open the instrument lid and remove the protective cap covering where the cuvette will be placed. Load the cuvette into the instrument in the correct orientation with the notch of the insert facing the camera and replace the cap and instrument lid. Click the streaming arrow to turn on the camera and click the chevron arrow to expand the record settings.Adjust the focus until the relatively small particles are clearly visible. To set the analysis for a small EV quantification, set the frame rate to 30 frames per second, the exposure to 15 milliseconds, the stir time to five seconds, the wait time to three seconds, the blue, green, and red laser powers to 210, 12, and 8 milliwatts respectively, the frames per video to 300, and the gain to 30 decibels. Adjust the focus until the relatively small particles are clearly visible.Increasing the zoom and/or the gain can help with particle focusing. But if you increase the gain, remember to set it to 30 decibels prior to recording. Once particles are in focus, set the zoom setting to 0.5X to save bandwidth and to prevent loss frames and click record to begin recording the video.When a prompt appears stating that the videos have been recorded, click OK to complete the recording and select the process tab. If very large particles were visible in any video while recording, navigate to the directory of recorded videos and remove any problematic video prior to processing. Check the disable audio detection override box and set the feature diameter to 30.Click process to initiate video processing and view a live distribution graph. When the processing is complete, click OK and select the plot tab. For EVs, display the main chart as log bin silica.Other features of the graph, such as changing the x-axis to set the area for integration of the figure produced, can be customized. To create a PDF report of the results, click the report button. The mean, median, mode size, and concentration adjusted for the dilution factor and distribution width will be displayed.NTA of the diluent should be performed before any sample so that the concentration of this blank can be subtracted from the EV sample particle concentration. To clean the cuvettes after analysis, empty the cuvette and completely fill the cuvettes 10 to 15 times with deionized water and the three times with 70 to 100%ethanol to remove any residual sample. Dry the outside of the cuvettes with a lint-free microfiber cloth and dry the inside with a compressed air duster.To clean the inserts and stir bars, place the materials in a glass scintillation vial containing 70 to 100%ethanol and shake the vial vigorously. Then rinse the inserts and stir bars in deionized water with shaking as demonstrated and dry them using lint-free cloths. After drying, immediately place all of the clean components into storage until the next analysis.Before performing an analysis, the instrument calibration was tested using polystyrene beads to ensure the validity of the acquired data. As observed, the particle tracking instrument accurately reported the size of the 100 nanometer manodisperse beads, but only closely reported the size of the 400 nanometer beads. Therefore, the instrument settings for this protocol were more accurate for smaller particles, closer to 100 nanometers in size.Using these settings, reported particle concentration scales accordingly with the dilution factor demonstrating that the instrument can accurately detect the particle concentration at various dilutions with little variability between technical replicants. The optimum dilution for a 4.41 times 10 to the 10th particles per milliliter mouse tissue-derived EV sample was determined to be between 1, 000 and 3, 000. In this analysis, increasing the gain increased the sensitivity of the camera, allowing an increase in the visualization of a higher number of smaller particles.Increasing the blue laser power from 70 to 210 milliwatts while keeping the green and red laser powers constant shifted the reported average particle size from 122 to 105 nanometers and increased the reported total particle concentration from 1.1 times 10 to the eighth to 1.7 times 10 to the eighth. Increasing the power of the red laser increased the reported average particle size from 175 to 246 nanometers and decreased the reported total particle concentration. Increasing the green laser power resulted in a decrease in the reported average particle size and an increase in reported total particle concentration.Finding the right dilution to place a sample within the optimal detection range can take a few tries for each sample. The cuvette cleaning also requires extra careful handling. We recommend applying more than one orthogonal method for EV particle size and concentration measurements.Dynamic light scattering, resistive pulse sensing, transmission electron microscopy, and single-particle interferometric reflectance imaging sensing can also be performed to characterize EVs.

nanoparticle-tracking-analysis-for-quantification-size-determination

Research

JoVE Journal

Biology

7.7K vues.  Columbia University Mailman School of Public Health. Les résultats de la NTA sont très sujets au biais de l’opérateur. Ce protocole démontre les effets de la modification des paramètres de l’ATN sur les résultats obtenus. Une méthode normalisée aidera à accroître la rigueur et la reproductibilité dans l’analyse NTA.L’analyse de l’échantillon dans une cuvette permet de capturer un échantillon statistiquement aléatoire dans chaque vidéo. Il en résulte des données plus reproductibles et la visualisation de particu...