Name: improving reproducibility to meet minimal information for studies of extracellular vesicles 2018 guidelines in nanoparticle tracking analysis
Uploaded: 2021-11-17T13:00:00
Description: Watch this Scientific Journal Video about Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis at JoVE.com

The work was supported by the state of Kansas to the Midwest Institute for Comparative Stem Cell Biology (MICSCB),&#160;the Johnson Cancer Research Center to MLW and NIH&#160;R21AG066488 to&#160;LKC. OLS received GRA support from the MICSCB. The authors thank Dr. Santosh Aryal for providing the liposomes used in this project and the members of the Weiss and Christenson laboratories for helpful conversations and feedback. Dr. Hong He is thanked for technical support. MLW thanks Betti Goren Weiss for her support and counsel.

1. General protocol guidelines
<ol>
	<li>Maintain the microscope on an air table or at a minimum on a vibration-free table. Ensure that extraneous vibrations (e.g., foot tapping on the floor, touching the table, door closures, laboratory traffic) are kept to a minimum.</li>
	<li>Set and maintain the temperature of the laser module at a constant temperature for all video recordings. 
	NOTE: The temperature chosen was 25 &#176;C because the nanoparticle size analyzer was calibrated at that temperatu...

<ol>
	<li>Lotvall, 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=Minimal+experimental+requirements+for+definition+of+extracellular+vesicles+and+their+functions:+a+position+statement+from+the+International+Society+for+Extracellular+Vesicles.">Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles.</a> Journal of Extracellular Vesicles. 3 (1), (2014).</li><li>Thery, C., 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=Minimal+information+for+studies+of+extracellular+vesicles+2018+(MISEV2018):+a+position+statement+of+the+International+Society+for+Extracellular+Vesicles+and+update+of+the+MISEV2014+guidelines.">Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.</a> Journal of Extracellular Vesicles. 7 (1), 1535750 (2018).</li><li>Consortium, E. -. T., 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=EV-TRACK:+transparent+reporting+and+centralizing+knowledge+in+extracellular+vesicle+research.">EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research.</a> Nature Methods. 14 (3), 228-232 (2017).</li><li>Gardiner, C., 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=Techniques+used+for+the+isolation+and+characterization+of+extracellular+vesicles:+results+of+a+worldwide+survey.">Techniques used for the isolation and characterization of extracellular vesicles: results of a worldwide survey.</a> Journal of Extracellular Vesicles. 5, 32945 (2016).</li><li>Maas, S. 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=Possibilities+and+limitations+of+current+technologies+for+quantification+of+biological+extracellular+vesicles+and+synthetic+mimics.">Possibilities and limitations of current technologies for quantification of biological extracellular vesicles and synthetic mimics.</a> Journal of Controlled Release. 200, 87-96 (2015).</li><li>Danaei, 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=Impact+of+particle+size+and+polydispersity+index+on+the+clinical+applications+of+lipidic+nanocarrier+systems.">Impact of particle size and polydispersity index on the clinical applications of lipidic nanocarrier systems.</a> Pharmaceutics. 10 (2), 57 (2018).</li><li>Kestens, V., Bozatzidis, V., De Temmerman, P. J., Ramaye, Y., Roebben, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+particle+tracking+analysis+method+for+the+size+determination+of+nano-+and+microparticles.">Validation of a particle tracking analysis method for the size determination of nano- and microparticles.</a> Journal of Nanoparticle Research. 19 (8), 271 (2017).</li><li>Filipe, V., Hawe, A., Jiskoot, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+evaluation+of+nanoparticle+tracking+analysis+(NTA)+by+NanoSight+for+the+measurement+of+nanoparticles+and+protein+aggregates.">Critical evaluation of nanoparticle tracking analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates.</a> Pharmaceutical Research. 27 (5), 796-810 (2010).</li><li>Hole, 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=Interlaboratory+comparison+of+size+measurements+on+nanoparticles+using+nanoparticle+tracking+analysis+(NTA).">Interlaboratory comparison of size measurements on nanoparticles using nanoparticle tracking analysis (NTA).</a> Journal of Nanoparticle Research. 15 (12), 2101 (2013).</li><li>Malvern analytical Ltd. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> NanoSight LM10 Operating Manual-P550H. , (2013).</li><li>Kim, A., Ng, W. B., Bernt, W., Cho, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+size+estimation+of+nanoparticle+tracking+analysis+on+polydisperse+macromolecule+assembly.">Validation of size estimation of nanoparticle tracking analysis on polydisperse macromolecule assembly.</a> Scientific Reports. 9 (1), 2639 (2019).</li><li>Gollwitzer, C., 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+comparison+of+techniques+for+size+measurement+of+nanoparticles+in+cell+culture+medium.">A comparison of techniques for size measurement of nanoparticles in cell culture medium.</a> Analytical Methods. 8 (26), 5272-5282 (2016).</li><li>vander Pol, 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=Particle+size+distribution+of+exosomes+and+microvesicles+determined+by+transmission+electron+microscopy,+flow+cytometry,+nanoparticle+tracking+analysis,+and+resistive+pulse+sensing.">Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing.</a> Journal of Thrombosis and Haemostasis. 12 (7), 1182-1192 (2014).</li></ol>

There are several methods available to estimate the size and concentration of nanoparticles11. These include ensemble methods that generate a size estimate from a population, including dynamic light scattering (DLS), centrifugal sedimentation, and single-particle level analysis-electron microscopy, NTA, atomic force microscopy, and tunable resistive pulse sensing. Of these, DLS and NTA are widely used, nondestructive size and concentration measurement methods, based on Brownian movement in an idea...

Accurate and repeatable analysis of EVs and other nanometer-scaled particles presents numerous challenges across research and industry. Replication of EV research has been difficult, in part, due to the lack of uniformity in reporting necessary parameters associated with data collection. To address these deficiencies, the ISEV proposed industry guidelines as a minimal set of biochemical, biophysical, and functional standards for EV researchers and published them as a position statement, commonly referred to as MISEV20141. The accelerating pace of EV research required an updated guideline, and the &#34;MISEV2018: a position statement of the ISEV&#34;&#160;expanded the MISEV2014 guidelines2. The MISEV2018 paper included tables, outlines of suggested protocols, and steps to follow to document specific EV-associated characterization. As a further measure to facilitate interpretation and replication of experiments, EV-TRACK was developed as a crowd-sourcing knowledgebase (http://evtrack.org) to enable more transparent reporting of EV biology and the methodology used for published results3. Despite these recommendations for standardized reporting of methods, the field continues to suffer regarding replicating and confirming published results.
Fitting with the National Institutes of Health&#39;s and National Science Foundation&#39;s effort for quality assessment tools, this paper suggests that ISEV requires standardized reporting of methods and details so that data assessment tools might be applied with the goal of replicating results between laboratories. Reporting cell sources, cell culture procedures, and EV isolation methods are important factors to define the qualities of the EV population. Among NTA instruments, factors such as detection settings, the refractive index of carrier fluid, heterogeneous particle populations contributing to polydispersity, lack of standardized reporting requirements, and absent intra- and inter-observer measurement results make NTA comparison between labs difficult or impossible.
In use since 2006, NTA is a popular method for nanoparticle size and concentration determination that is currently used by approximately 80% of EV researchers4. The MISEV2018 Guidelines require two forms of single-vesicle analysis, of which NTA is one of the popular options. NTA continues to be in common use for EV characterization due to its wide accessibility, low cost per sample, and its straightforward founding theory (the Stokes-Einstein equation). EV assessment by NTA generates a particle size distribution and concentration estimate using laser light scattering and Brownian motion analysis, with the lower limit of detection determined by the refractive index of the EV. When using a fluid sample of known viscosity and temperature, the trajectories of the EVs are tracked to determine their mean-square displacement in two dimensions. This allows the particle diffusion coefficient to be calculated and converted into a sphere-equivalent hydrodynamic diameter by a modified Stokes-Einstein equation5,6,7. NTA&#39;s particle-to-particle analysis has less interference by agglomerates or larger particles in a heterogeneous population of EVs than other methods of characterization7. While a few larger particles have minimal impact on sizing accuracy, the presence of even minute amounts of large, high light-scattering particles results in a notable reduction in the detection of smaller particles due to reduced software EV detection and tracking8. As a measurement technique, NTA is generally considered not to be biased toward larger particles or aggregates of particles but can resolve multiple-sized populations through individual particle analysis9. Because of the use of light-scattering by particles, one of the limitations of NTA analysis is that any particulate such as dust, plastic, or powder with similar refraction and size attributes compared to EVs cannot be differentiated from actual EVs by this method of characterization.
The NanoSight LM10 (nanoparticle size analyzer) and LM14 (laser module) have been sold since 2006, and although newer models of this instrument have been developed, this particular model is found in many core facilities and is considered a reliable workhorse. Training is needed to properly optimize the NTA settings for high-resolution measurements of size and concentration. The two important settings needed for optimum video recordings are (1) the camera level and (2) the detection threshold. These must be set by the operator based on the sample&#39;s characteristics. One of the major constraints of NTA analysis is the recommendation of sample concentrations between 107 and 109 particles/mL, to achieve this sample dilution may be required10. Solutions used for dilution, such as phosphate-buffered saline, 0.15 M saline, or ultrapure water, are rarely free of particles less than 220 &#181;m in size, which may affect the NTA measurements. NTA characterization of the solutions used for dilution should be performed at the same camera level and detection threshold as the nanoparticle samples that are being analyzed.The size and concentration of nanoparticles present in diluents used for EV sample dilutions are seldom included in publications involving NTA analysis of EVs.
This protocol uses NTA analysis of synthetic EV-like liposomes evaluated using selected camera levels, detection thresholds, and mechanical filtering of the samples to analyze the systematic effects of camera level, detection threshold, or sample filtration on the NTA dataset. Liposomes were synthesized as described in Supplemental File&#160;S1. Synthetic liposomes were used in this experiment because of their size uniformity, physical characteristics, and stability in storage at 4 &#176;C. Although actual samples of EVs could have been used, the heterogenicity and stability of EVs during storage may have complicated this study and its interpretation. Similarities in the NTA reports from (A) liposomes and (B) EVs indicate that the systematic effects revealed for liposomes in this paper will likely also apply to EV characterization (Figure 1). Together, these findings support the notion that complete reporting of critical software settings and the description of sample processing, such as diluent, dilution, and filtration, impact the reproducibility of NTA data.
The purpose of this paper is to demonstrate that varying the NTA settings (temperature, camera level, and detection threshold) and sample preparation changes the results collected: systematic, significant differences in size and concentration were obtained. As NTA is one of the popular options to fulfill the MISEV2018 characterization specification, these results demonstrate the importance of reporting sample preparation and NTA settings to ensure reproducibility.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63059/63059fig01.jpg" /> 
Figure 1: Representative NTA reports to compare liposomes to EVs. (A) Liposomes: unfiltered sample characterized on NTA on 12 March 2020. (B) EVs: unfiltered sample characterized on NTA on 26 August 2021. Abbreviations: NTA = Nanoparticle tracking analysis; EVs = extracellular vesicles. <a href="https://www.jove.com/files/ftp_upload/63059/63059fig01large.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>Automatic Pipetter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes, Conical, Nunc 15 mL</td>
			<td>Thermo Sci.</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lens Cleaner</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lens Paper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoSight LM-10</td>
			<td>Malvern Panalytical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoSight LM-14 Laser Module</td>
			<td>Malvern Panalytical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanosight NTA Software Ver. 3.2</td>
			<td>Malvern Panalytical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paper Towels</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips, 1-200 &#181;L, Filtered, Sterile, Low Binding</td>
			<td>BioExpress</td>
			<td>P -3243-200X</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips, 50-1,000 &#181;L, Filtered, Sterile</td>
			<td>BioExpress</td>
			<td>P-3243-1250</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline, Dulbecco&#39;s Phosphate Buffered (No Ca or Mg)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Standards, Latex Transfer- 100 nm (3 mL)</td>
			<td>Malvern</td>
			<td>NTA4088</td>
			<td></td>
		</tr>
		<tr>
			<td>Standards, Latex Transfer- 50 nm&#160; (3 mL)</td>
			<td>Malvern</td>
			<td>NTA4087</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filter, 33 mm, .22 &#181;m, MCE, Sterile</td>
			<td>Fisher brand</td>
			<td>09-720-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, TB, 1 mL, slip tip</td>
			<td>Becton Dickinson</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Waste fluid container</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

improving reproducibility to meet minimal information for studies of extracellular vesicles 2018 guidelines in nanoparticle tracking analysis

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many consider that NTA instruments and their software packages can be easily utilized following minimal training and that size calibration is feasible in-house. As both NTA acquisition and software analysis constitute EV characterization, they are addressed in Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018). In addition, they have been monitored by Transparent Reporting and Centralizing Knowledge in Extracellular Vesicle Research (EV-TRACK) to improve the robustness of EV experiments (e.g., minimize experimental variation due to uncontrolled factors).
Despite efforts to encourage the reporting of methods and controls, many published research papers fail to report critical settings needed to reproduce the original NTA observations. Few papers report the NTA characterization of negative controls or diluents, evidently assuming that commercially available products, such as phosphate-buffered saline or ultrapure distilled water, are particulate-free. Similarly, positive controls or size standards are seldom reported by researchers to verify particle sizing. The Stokes-Einstein equation incorporates sample viscosity and temperature variables to determine particle displacement. Reporting the stable laser chamber temperature during the entire sample video collection is, therefore, an essential control measure for accurate replication. The filtration of samples or diluents is also not routinely reported, and if so, the specifics of the filter (manufacturer, membrane material, pore size) and storage conditions are seldom included. The International Society for Extracellular Vesicle (ISEV)&#39;s minimal standards of acceptable experimental detail should include a well-documented NTA protocol for the characterization of EVs. The following experiment provides evidence that an NTA analysis protocol needs to be established by the individual researcher and included in the methods of publications that use NTA characterization as one of the options to fulfill MISEV2018 requirements for single vesicle characterization.

Table 1 contains the results of the NTA videos for the liposome samples (18 filtered and 18 unfiltered) and a representative DPBS diluent. Comparisons across the two groups were completed regardless of the camera level or detection threshold in this paper. Filtered samples had a mean particle diameter of 108.5 nm, a particle mode of 86.2 nm, and a concentration of 7.4 &#215; 108 particles/mL. In contrast, unfiltered samples had a mean particle diameter of 159.1 nm, a particle mode of 105.7 nm,...

Watch this Scientific Journal Video about Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis at JoVE.com

Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis

Nanoparticle tracking analysis (NTA) is a widely used method to characterize extracellular vesicles. This paper highlights NTA experimental parameters and controls plus a uniform method of analysis and characterization of samples and diluents necessary to supplement the guidelines proposed by MISEV2018 and EV-TRACK for reproducibility between laboratories.

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many ...

This protocol was developed for novice operators of the NanoSight LM10. By following the step-by-step instructions, accurate and reproducible results can be obtained. This technique helps ensure consistent results across multiple trial runs regardless of the user's skill level.Nanoparticle characterization, especially quantification, continues to be a challenge in the extracellular vesicle research field. This protocol allows a consistent size and concentration analysis of nanoparticles in suspension. Begin by cleaning the glass surfaces of the laser module with a good quality lens cleaner and lens paper.Prior to assembly of the module, make sure that the O-ring seal is properly seated in the groove of the flow cell cover. Then place the flow cell cover on the laser module, ensuring that electrical contacts are in the proper orientation. Next, place the four spring-loaded thumb screws through the flow cell plate and engage the threads of the laser module without tightening individually.While putting uniform pressure down on the flow cell cover, uniformly tighten the thumb screws in an alternating diagonal manner until snug. Flush two one milliliter tuberculin syringes with slip lock adapters three times with one milliliter of DPBS. Remove and discard the plunger from the first tuberculin syringe before inserting the syringe into the remaining port to serve as a voided diluent or sample reservoir.Then fill the second syringe with one milliliter of DPBS and attach it to the inlet port of the flow cell cover. Hold the laser module tilted with the outlet syringe port elevated to allow air to be purged from the chamber as the DPBS is injected slowly into the laser module. Flush the remaining DPBS from the laser module by injecting one milliliter of air into the inlet port.Repeat flushing twice. After the last flush, empty the laser module as completely as possible and load the module for focusing and positioning. Open the software.Under the capture tab in the upper left corner box, click start camera. If the camera shuts off automatically after five minutes, click start camera to restart it. In the same tab, adjust camera level to 14 to 16 to brighten the laser line and simplify particle identification and focus.By moving the top slider on the left side of the head piece in or out, divert the image from the camera to the eyepieces. In the field of view, find the area of increased density, referred to as the thumbprint and center, and focus the thumbprint vertically. In the field of view, center the laser line, then move the top slider to divert light to the camera as observed on the computer screen.The image on the computer screen is a mirror image from the view in the microscope eyepieces. Adjust the focus to sharpen the image of individual moving particles on the screen with the focus knob. To load the samples, draw up one milliliter of the sample into a rinsed one milliliter tuberculin syringe and attach the syringe to the inlet port of the flow cell cover.Keep advancing the plunger until fluid is evident in the open syringe attached to the outlet port. The module should be tilted to allow air to be purged from the laser module. In the camera view, move the focus to the right of the laser line to an area of a uniform number of particles.Adjust the vertical orientation to center the horizontal bands of light and refocus until the highest number of particles are in view. Ensure to maintain the position of the focus consistent for all subsequent measures. Adjust the camera level to the point that the dark information symbol flashes intermittently on and off in the top right of the camera view.For nanoparticle tracking analysis, or NTA, set the duration to 30 or 60 seconds and the number of videos to five. To change the existing base filename, click the tab for a new storage site for generated data with a new filename. Then check the target temperature box to input the desired temperature and click on create script to reuse the standard measurement.Once the sample is loaded and the experiment is ready to run, click create and run script. In the set report details pop-up screen, fill out the fields with the necessary information on the operator, sample, and diluent. When all desired fields have been filled, click settings OK to initiate the script.Prior to each video capture, look for a prompt to advance the plunger manually. Inject approximately 0.05 milliliters of the sample into the laser chamber. As the particles come to rest, click OK.Upon completing the fifth video capture, a settings confirmation box will appear along with the process box.As the frames are advanced manually from the bottom of the video screen, note the number of blue crosses making particles on the screen and set the detection threshold for processing of the sample. Wait for the videos to be automatically processed in a histogram of results before a dialog box notification of completion is displayed, then hit OK.After the export settings box appears, save the results by clicking export. Highlight all five of the capture videos listed in the current experiment.Next, click process selected files and wait for the setting confirmation box to appear in the process box to flash to change detection threshold, then adjust the detection threshold to the desired level and go to setting and click OK.The videos will be processed automatically in a histogram of results and a dialog box notification of completion will be displayed. Click OK.In the representative analysis, the results of the nanotracking analysis, or NTA, for the liposome samples and a representative DPBS diluent are shown. Filtered samples had a mean particle diameter of 108 nanometers and a concentration of 7.4 times 10 to the eighth particles per milliliter.In contrast, unfiltered samples had a mean particle diameter of 159 nanometers and a concentration of 7.6 times 10 to the eighth particles per milliliter. At combined camera levels, as the detection threshold was increased from two to five, mean and mode particle size showed a significant decrease in particle sizes of the filtered samples. Particle concentrations at combined camera levels were decreased as the detection threshold was increased.No significant difference in particle concentrations between filtered and unfiltered samples was detected. At combined detection thresholds, as the camera level increased from 12 to 14, a decrease in mean and mode particle size of the filtered samples was noted. Particle concentrations at combined detection thresholds increased as camera levels increased from 12 to 14.No significant differences between filtered and unfiltered samples was observed. Steps 4.1 through 5.2 are important to find the correct viewing area. Achieving consistent results across multiple trial runs depends on this repeatable skill.Dynamic light scattering is often used as a companion to nanoparticle tracking analysis primarily to get the zeta potential, which is the surface charge of the particles. For nanoparticle analysis, NTA continues to be a very valuable method of quantification of particles useful for extracellular vesicle research.

Research

JoVE Journal

Bioengineering

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

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A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

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Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

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3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

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Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

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Retroductal Nanoparticle Injection to the Murine Submandibular Gland

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Evaluation of the Storage Stability of Extracellular Vesicles

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A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

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Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins ...