In this protocol, we describe the complete workflow for rapid isolation of extracellular vesicles from human whole blood and characterization of specific markers by fluorescence-based nanoparticle-tracking analysis. The presented results show a high level of reproducibility and can be adjusted to cell culture supernatants.
This method addresses a common problem in the field and provides a complete workflow for rapid isolation and characterization of single extracellular vesicles with specific markers of interest. Nanoparticle-tracking analysis is a semi-automated method. It utilizes the properties of light-scattering and Brownian motion to analyze the size distribution of extracellular vesicles.
Demonstrating the procedure will be Vera Schmidt, a Ph.D.student from our laboratory. To isolate the extracellular vesicles, after collecting two milliliter samples of human whole blood into serum-separating tubes, allow the samples to coagulate for 15 minutes at room temperature before separating the cells from the serum by centrifugation. Transfer one milliliter of serum from each sample into individual 1.5 milliliter reaction tubes for centrifugation to remove the platelets, and transfer 100 microliters of the platelet-poor plasma to new 1.5 milliliter reaction tubes.
Next, add 25 microliters of exosome precipitation solution to the plasma and vortex the samples thoroughly. After a 30-minute incubation on ice, collect the extracellular vesicles by centrifugation. The pellet will appear as a beige or white color.
Aspirate the supernatant and centrifuge the sample again. Then, remove all trances of fluid and resuspend the pellet in 100 microliters of PBS. To stain the cell membranes of the vesicles, add 10 microliters of the EV suspension to 50 microliters of freshly-prepared PKH67 ethanolic dye solution and mix thoroughly.
After five minutes at room temperature in the dark, dilute 50 microliters of the stained extracellular vesicle suspension with 2.5 milliliters of distilled water in a 15 milliliter conical tube, with thorough mixing. To label the vesicles with antibodies, dilute 10 to 20 microliters of the unstained extracellular vesicle suspension with 50 microliters of distilled water in a 1.5 milliliter reaction tube, with thorough mixing. Add 2.5 to five microliters of the antibody of interest to the reaction tube, with thorough mixing, for a 30-minute incubation at room temperature in the dark.
Then, transfer 50 microliters of the labeled vesicles to a 15 milliliter conical tube containing the appropriate experimental volume of distilled water, with thorough mixing, as indicated in the table. Before analyzing the stained or labeled extracellular vesicles, first move the fluorescence filter into the optical path of the microscope and camera, and start the program, following the instructions on the screen for automated implementation. Under the Cell Check tab, select the correct Cell Number for fluorescence measurement and the reference position for the optics, to confirm that the laser and microscope are in a common focus.
Use a syringe to flush the cell channel with 10 milliliters of distilled water, taking care that the measurement cell is free of air bubbles and that air bubbles are not injected into the system. Next, dilute 10 microliters of 200-nanometer sized fluorescence-labeled polystyrene particles in 990 microliters of distilled water and add 10 microliters of this particle suspension into a 15 milliliter tube containing 10 milliliters of distilled water. Then, inject 2.5 milliliters of the first diluted particle solution into the cell channel and click Optimize Focus to adjust the camera.
To measure the sample, flush the cell channel multiple times with 10 milliliters of distilled water and inject the stained extracellular vesicle suspension. Using the reference position, adjust the appropriate acquisition parameters under the Cell Check tab, as indicated in the table. To identify the optimal sensitivity range, click Number of Particles vs.
Sensitivity, to display a curve of the measured particles per screen for the different sensitivity levels. For the Shutter parameter, adjust the period of time that the camera allows the light to pass for a determined interval. For the Postacquisition Parameters, select of a minimum brightness of 20, a minimum size of 20 nanometers, and a maximum measurement size of 500 nanometers.
Note the number of detected particles in the field of view of the display. The scattering bar should be in the green to orange, 50 to 300 particles range. Click Check Particle Drift at Zero Volts and open the Measurement tab.
Click Run Video Acquisition and set the Number of Experiments to three to five, and the Time Delay between experiments to zero minutes. Set the number of individual subvolume positions to 11 and the number of measurement cycles to 10 at each measurement position at which the particles are to be analyzed. Then, select a folder, create a new file name, and click Okay to start the measurement.
At the end of the analysis, open the Analysis tab to review the results. Then, check the average number of particles per position, the total number of traced particles and the particle concentration, the distribution width of the particles, and the value of the mean, and the standard deviation. Using fluorescent beads for the adjustment and calibration of the measurement gives an optimum setting sensitivity of 85%Using these settings, the camera displays a sharp picture and repeated measurements demonstrate a low standard deviation.
Staining with a cell linker kit that includes a fluorescent cell linker that incorporates a green fluorescent dye with long, aliphatic tails into lipid regions of the cell membrane, reveals a strong correlation between the sensitivity and the number of particles measured. Extracellular vesicle staining antibody against a protein of interest indicates a particle distribution from 220 to 1, 145 nanometers, with a peak maximum size of 541 nanometers. No extracellular vesicles are detected after antibody staining of control vesicle-free water, up to a sensitivity close to 100%If the measurement is started when the drift is greater than 5 micrometers per second, the individual repetitions demonstrate distinct deviations.
It is important to note that using fluorescein isothiocyanate as a fluorochrome results in inaccurate and unreproducible measurements, as this fluorophore is prone to rapid photobleaching. This protocol addresses the leading demand of many researchers in this field for a fast characterization of single extracellular vesicles, using specific markers. Despite the fact that, over the past decade, methods have improved considerably, there is yet no standardized protocol for analysis of single extracellular vesicles.
Irrespective of future progress of research on extracellular vesicle biology, this method provides a rapid and reliable protocol for characterization of single extracellular vesicles using specific markers. Because our current protocols do not involve long processing times or labor-intensive steps, they are also suitable for high sample throughput.
