Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

This protocol provides a rapid and size-specific solution method for small extracellular vesicles by optimizing flow cytometry sorting parameters. These optimizations provide a panel of critical sort settings that enables one to obtain representative populations of small extracellular vesicles using forward scatter versus size scatter only. Begin by performing the standard startup procedure of the flow cell sorter.

Turn on the machine by pressing the startup button, then click the laser power button on the laser control panel. Press the change tanks button on the Smart Sampler submenu to pressurize the fluidics. Use an ultrasonically cleaned 50 micrometer jet and air nozzle and install it down to the nozzle assembly.

Adjust the pressure to 80 SI for sheath fluid and 80.3 SI for sample flow on the pressure console. Press the start sheath flow button to start the sheath stream flowing. Click the bubble button in the Smart Sampler submenu to automatically debubble for 10 minutes and then turn it off.

To align the stream and determine the laser spot, turn on the illumination chamber light to view the stream over the pinholes. Adjust the up and down, left and right, and front and back micrometers to center the stream on the pinhole and focus the stream. Open all laser shutters, select the laser and stream intercept tab and then press the green arrow button continuously as prompted to complete the laser intercept and nozzle alignment calibration process.

Access the fine laser alignment screen. Select the 640 nanometer laser and 772 bar 44 bandpass channel as the x-axis parameter and the 405 nanometer laser and 448 bar 59 bandpass channel as the y-axis parameter. Press the trigger parameter selector and choose the forward scatter parameter of the 488 nanometer laser.

Next, dilute the rainbow fluorescent particles by adding one drop into one milliliter of double distilled water to a final concentration of one times 10 to the sixth beads per milliliter. Open the sample chamber door and load a tube of prepared rainbow fluorescent particles. Press the start sample button and adjust up and down micrometers to optimize fluorescence intensity, making each signal as intense as possible.

Press the start quality control or QC button on the touchscreen control panel to perform QC automatically. Then press the IntelliSort initialization button. The instrument automatically adjusts the frequency and amplitude of droplet oscillation to obtain the drop delay of approximately 25 and be able to visualize the break off point of the drop clearly.

Next, press the drop delay button and the maintain button. Select tube as the sort output type. Place a 15 milliliter tube holder in the sort chamber.

Turn the plate voltage on and set the plate voltage to approximately 4, 000 volts. Next, turn on the test pattern by selecting the stream setup button. Adjust the charge phase slider and deflection slider to ensure that the image of the stream is in line with the collection tube.

Then select the check mark button. Log in to the Summit workstation on the computer. Create a new protocol from the file main menu.

Then create a dot plot by selecting the histogram tab in the Summit Software control panel. Choose forward scatter as the x-axis parameter and side scatter as the y-axis parameter. Then right-click on the axis of the new dot plot in the workspace and present the forward scatter and side scatter in the logarithmic form.

Select the acquisition tab and locate the acquisition parameters panel. Enable all signals in the submenu of enabling parameters. Choose forward scatter as the trigger parameter and set the threshold of 0.01.

Adjust the voltage and gain of forward scatter and side scatter at 250 and 0.6 to ensure events within the forward scatter versus side scatter plot and populations are separated. Dilute the fluorescent polystyrene microspheres into 100, 200, and 300 nanometers suspensions by adding one microliter of microspheres to one milliliter of double distilled water. Then load the microsphere suspension successively.

Select Start under the acquisition menu of Summit Software to record 20, 000 events and then click Stop after recording the events. Right-click on the forward scatter versus side scatter dot plot to create rectangles and drag to resize. Reposition the regions of electronic noise and 100 nanometer microsphere population by right-clicking on the regions to rename them as R7 and R4 respectively.

Repeat the steps to frame the 200 and 300 nanometer microspheres with rectangles successively, and rename them as R5 and R6 respectively. Finally, determine the 50 to 200 nanometer sorting region based on the electronic noise and the position of 200 nanometer particles to define it as R8.Edit sort decisions in the sort logic and statistics panel by double clicking on the blank field of the left one or L1 stream and selecting the region named R8 to create the sort logic. Choose the abort mode of purity and select a droplet envelope above one drop for the L1 stream.

Load 500 microliter sample of previously prepared extracellular vesicles or EVs mixture. Press the Start button under the acquisition menu in Summit to acquire the data and then click Stop. Next, press Start in the sort menu to collect 50 to 200 nanometer sized small extracellular vesicles or sEV into a 15 milliliter centrifuge tube.

Flow cytometry analysis of standard-sized polystyrene microspheres to locate the 50 to 200 nanometer size range in the forward scatter versus side scatter plot revealed distinguishable particle signals. R4, R5, and R6 refer to the positions of 100, 200, and 300 nanometer particles respectively. R7 is the electronic noise as the detection limit for 50 nanometer particles and R8 represents the position range of 50 to 200 nanometer particles.

The particle size distribution of the EVs mixture derived from pancreatic one cells by nanoparticle tracking analysis, or NTA, was in the wide range of 40 to 400 nanometers after ultra centrifugation. Flow cytometric sorting was performed to isolate and purify the 50 to 200 nanometer sEV within the R8 region. The quality of isolation was verified by NTA and it was found that the particle size range of sEV after sorting was between 50 to 200 nanometers.

The presence of sEV was further observed by TEM and the isolated sEVs were confirmed to contain markers of CD6, CD63, and CD81 by Western Blot Analysis. This method allows for the separation of small extracellular vesicles and the classification or pro tem analysis of small extracellular vesicles gene expression opening up numerous downstream research applications.