Name: nanoparticle tracking analysis for the quantification and size determination of extracellular vesicles
Uploaded: 2021-03-28T14:00:00
Description: Watch this Scientific Journal Video about Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles at JoVE.com

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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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 ...

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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 ...

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

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 ...