The goal of this procedure is to analyze exosomes by their size and to quantify them by using a nanoparticle tracking analysis or NTA instrument through a semi-automated method. This is accomplished by using differential centrification steps to obtain an exosome pellet from human blood. The exosomes are resuspended at a concentration that is predetermined for the amount of starting plasma to obtain the appropriate scatter intensity.
During NTA, the suspension is then filtered to separate exosomes from larger particles. Next, a calibration of the NTA instrument is performed using a control suspension containing 200 nanometer sized polystyrene particles. The exosome solution is then injected into the NTA instrument and the ideal setting of the NTA is selected by performing a pretest before performing the final test.
Ultimately, results can be obtained that show quantification of all measured particles and listing of the particles by their sizes. The demonstration of this method is critical as the solution and the setting for each prepared exosome solution has to be performed individually to gain the best results. The measured exosomes should be in an I concentration so that the live view mode displays around 600 particles per screen.
Also, a pretest should be performed to find the optimal sensitivity range for the measurement. A high setting of sensitivity leads to visualization of more small particles, would also increases issues related background noises. The procedure will be demonstrated by Anto May, a grad student from our laboratory To begin exosome preparation, collect whole blood in three citrate tubes via venipuncture for a total of nine milliliters.
Then pour the blood into a 15 milliliter Falcon tube centrifuge the sample at 1, 500 G for 20 minutes of four degrees Celsius to initiate separation of cells from plasma. Transfer the S nascent to a new 15 milliliter Falcon tube. Then centrifuge the sample again at 2, 800 G for 20 minutes at four degrees Celsius to remove all cells from the plasma.
Transfer the cell-free plasma or CFP to ultracentrifugation tubes at one milliliter per tube. Next centrifuge the CFP at 100, 000 G for 90 minutes at four degrees Celsius to deplete exosomes. Remove 900 microliters of supernatant and resuspend the pellet in the remaining 100 microliters in the ultracentrifugation tube.
After adding 900 microliters of PBS centrifuge, you gain at 100, 000 G for 30 minutes at four degrees Celsius. As before, remove 900 microliters of the supine agent and resuspend the pellets with the remaining 100 microliters. Transfer five to 20 microliters of the Resus suspension to 40 milliliters of distilled water.
Filter the suspension through a 450 nanometer filter to separate exosomes from larger particles. Use this final suspension for particle measurement to use the particle tracking instrument. First, start the program by double clicking on the software icon.
Click on the various software tabs to switch between them throughout the protocol. Follow the instructions on the screen. For an automated implementation of the startup procedure, select the boxes for both cell quality check and auto alignment.
Either of these steps can be repeated separately if necessary by pressing button A or B on the cell check tab. Place a beaker under the outlook port to collect waste solution and do not inject air bubbles into the system. Open the inlet and outlook port of the nanoparticle tracking analysis or NTA instrument and inject 10 milliliters of distilled water by syringe into the cell channel through the inlet port.
Close the inlet and outlet port immediately as the last amount of water is being injected. Ensure that the measurement cell is free of air bubbles. Perform the cell quality check by clicking on.Okay.
The software will display the cell quality result after a few seconds, if any particles are still visualized in the live view screen of the software, or if the result of the quality check is only good or poor, repeat the distal water injection until the remaining particles are removed from the measurement cell L.Also, repeat this step after each measurement to avoid accumulation of particles. Next, prepare a control suspension containing uniform, 200 nanometer sized polystyrene particles. To align the foci of the laser and microscope, add one drop of the suspension concentrate provided by the instrument manufacturer to 500 milliliters of distilled water resulting in the required concentration such that 600 plus or minus 100 particles are displayed per screen in the live view.
Inject the alignment suspension into the NTA instrument as done for the distilled water press. Okay to start also alignment, which is an automated routine through which the system will automatically find the optimum position of the two foci. When a prompt appears stating the system is now ready for experiments, click on okay to start the measurement.
Occasionally clean the cell channel manually between experiments by flushing the cell with a 30%solution of ethanol. Clean the channel whenever the software displays an automatic error report. To perform measurement of the system.
First, flush the channel with distilled water prior to each sample measurement. Then inject the exosome suspension into the channel. The next step is to adjust the main parameters in the software to adjust the sensitivity.
Find the optimal sensitivity range by clicking on number of particles versus sensitivity to display a curve for measured particles per screen for different sensitivity levels. Choose a sensitivity level before the maximum slope of the curve. A higher sensitivity level allows visualization of more small particles, but also increases issues related to background noise.
To adjust minimum brightness, choose a starting brightness of 20 for exosome measurement. To use this function as a filter, regulate this parameter up to blank out strongly light scattering particles. Regulate it down to amplify weekly scattering artifacts, very brightness as needed throughout the experiment.
Next, set a digital filter to eliminate pixel noise and unwanted scatter oversized particles. By regulating the minimum and maximum size, use a range of 10 to 500 pixels for measurement of exosomes. Adjust the shutter by changing the period of time that the camera is open by setting shutter to 300, which is equal to 3.3 milliseconds.
To adjust the live readout parameters switch between a digital and analog view modus at any point by clicking on the live image button. Throughout the experiment. Monitor the scattering intensity bar, which displays the saturation status of the sample as a color coded indicator.
Do not analyze exosomes if the scattering bar is red. To begin measurement, click on the measurement tab. Choose settings in the run options array.
Before each acquisition, select check movement, a repeated check particle drift test, select the number of experiments and the time delay between them. Perform a sample check if necessary. Finally, click on the run video acquisition button.
In the new window, define the number of cycles and the number of measurement positions. Confirm the minimal time duration. A particle is tracked to be counted as one individual particle.
By setting the video resolution, a lower resolution results in a shorter tracking duration. Finally, select a file name and click on okay to start measurement. The number of particles versus sensitivity curve displays the particles in one position at one moment.
During an automatic sensitivity scan, a sensitivity value is chosen prior to the maximum slope of the graph. Artifacts are not eliminated in this test experiment and can affect the graph. The visualization of particles on the live view screen is helpful To set the right sensitivity value.
A value that is too low leads to detection of few particles. While a value that is too high shows a poor image, quality particles appear a single dots well isolated from each other in the optimum sensitivity level. The result tab after measurement allows the readout of parameters, including total number of particles traced average number of particles per position and particle concentration, as well as the numerical ratio of particle size to the percentage of particle numbers.
The graph calculated after the measurement shows the distribution of particles by size. Icons in the display mode can be used to change the settings of the graph. Also, there are different ways to analyze exosomes.
This method shows a very simple and elegant method to perform a similar method analysis in a fast process to generate results in a short time. In order to compare multiple samples, we recommend keeping the same dilution level for all samples. All settings accept the sensitivity and the video resolution can be changed afterwards by using the set of few analysis software.
This way, comparative analysis of data obtained in different experiments on exosome quantity and size becomes available in the semi-automated fashion.
