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

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.