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

1. Cell culture

1. Prepare a culture medium of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Culture the human pancreatic cancer cell, PANC-1, in a 75 cm² culture flask at a density of 1 x 10⁶ cells/mL. Incubate the culture at 37 °C with 5% CO₂.
2. Subculture the cells when cell density reaches 75%-80% under microscopic observation. Remove the medium, and rinse 2x with 3-5 mL of phosphate buffer saline (PBS; 0.01 M, pH 7.2-7.4).
3. Discard the solution, add 1-2 mL of trypsin-EDTA solution, and let the cells detach until they become rounded and suspended in the medium under the microscope. Add fresh medium, aspirate, and disperse into 75 cm² culture flasks with 15 mL culture medium. Use a sub-cultivation ratio of 1:2 to 1:4 (recommended).
   ​NOTE: All operations are performed in a biosafety cabinet to avoid cell contamination.

2. Culture medium collection

1. Incubate the cells for 48 h at 37 °C with 5% CO₂. Collect cell supernatant (90 mL) in two 50 mL centrifuge tubes and centrifuge at 300 x g for 10 min at 4 °C.
2. Transfer the supernatant to new sterile tubes and centrifuge successively at 2,000 x g for 20 min, 10 000 x g for 30 min, and 12,000 x g for 70 min at 4 °C.
3. Remove the supernatant and rinse the pellet with 10 mL of PBS by pipetting gently. Centrifuge at 12,000 x g for 70 min at 4 °C again and resuspend the pellet in 10 mL of PBS buffer to obtain a mixture of EVs.
4. Perform nanoparticle tracking analysis (NTA) to quantify and size the EV mixture. Perform the NTA analysis according to the instrument operation reference manual and reference²¹.

3. Cell sorting

1. Perform the standard startup procedure of the flow cell sorter. Turn ON the machine by pressing the Startup button. Click the Laser Power button on the laser control panel. Press the Change Tanks button in the smart sampler submenu to pressurize the fluidics.
   NOTE: Ensure that the waste tank is empty, and the sheath tank is full before starting up.
2. Use an ultrasonically cleaned 50 µm jet-in-air nozzle and install it onto the nozzle assembly. Adjust the pressure to 80 psi for sheath fluid and 80.3 psi for sample flow on the pressure console.
3. 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 min and then turn it off.
   NOTE: For cell sorting, different sizes of air spray nozzles require different sheath fluid pressures to maintain liquid flow stability. In this method, a nozzle of 50 µm was used, and only a sheath fluid pressure greater than or equal to 80 psi could generate stable side streams. The pressure difference between the sample flow and the sheath fluid determines the loading speed of sorting. Under the setting condition of the method (the pressure difference between the sample flow and the sheath fluid is 0.3-0.6 psi), the liquid flow was relatively stable, and the loading speed enabled the sorting efficiency to increase to 80% from 50% for pressure difference less than 0.3-0.6 psi.
4. 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.
5. Select the Laser and Stream Intercept tab. Press the Green Arrow button continuously as prompted to complete the laser intercept and nozzle alignment calibration process.
6. Access the fine laser alignment screen. Select 640-722/44 (H) channel as the X-axis parameter and 405-448/59 (H) channel as the Y-axis parameter. Press the Trigger Parameter Selector and choose the SSC Parameter of the 488 nm laser. Open all laser shutters.
   NOTE: The X-axis and Y-axis parameters chosen depend on the laser configuration of the system. Other parameters are allowed if the system does not have a 640 nm or 405 nm laser. For best results, select lasers with the greatest spatial separation on the pinhole strip (not including the 355 nm laser).
7. Dilute the rainbow fluorescent particles by adding one drop into 1 mL of double distilled water for a final concentration of 1 x 10⁶ beads per mL. Open the door of the sample chamber and load a tube of the prepared rainbow fluorescent particles.
8. Press the Start Sample button. Adjust up-and-down micrometers to optimize fluorescence intensity, making each signal as intense as possible. Press the Start Quality Control (QC) button on the touchscreen control panel to perform QC automatically.
9. Perform drop delay. Access the sort setup screen and select the drop delay button. Press the IntelliSort Initialization button. The instrument automatically adjusts the frequency and amplitude of droplet oscillation to obtain the drop delay of 25 ± 5 and be able to visualize the break-off point of the drop clearly. Press the Maintain button.
10. Select tube as the sort output type. Place a 15 mL tube holder in the sort chamber. Turn the plate voltage ON and set the plate voltage to approximately 4000 V.
11. 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 lined up with the collection tube. Select the Check Mark button.
12. Log in to the Summit workstation on the computer. Create a new protocol from the File main menu. Create a dot plot by selecting the Histogram tab in the Summit software control panel.
13. Right-click the axes of the new dot plot in the workspace and choose FSC as the X-axis parameter and SSC as the Y-axis parameter. Present the FSC and SSC in logarithmic form.
14. Select the Acquisition tab and locate the acquisition parameters panel. Enable all signals in the submenu of enabling parameters. Choose FSC as the trigger parameter and set the threshold of 0.01. Adjust the voltage and gain of FSC and SSC at 250 and 0.6 to ensure events within the FSC vs. SSC plot and populations are separated.
    ​NOTE: Threshold setting is equivalent to setting the particle size that can be detected by flow cytometry. The larger the threshold is, the larger the detected particles will be, and the small particles will be cut off by the threshold and cannot be detected. In this method, the threshold is set to 0.01, so all particles larger than electronic noise can be detected.

4. Isolation of sEV

1. Dilute the fluorescent polystyrene microspheres to suspensions of 100 nm, 200 nm, and 300 nm. Add 1 µL of microspheres to 1 mL of double distilled water.
2. Load the microsphere suspension successively. Select Start under the acquisition menu of summit software and record 20,000 events.
   NOTE: The number of events is optional. No less than 5,000 events are recommended to meet statistical significance.
3. Right-click on the FSC vs. SSC dot plot to create rectangles and drag to resize and reposition the regions of electronic noise and 100 nm microsphere population. Right-click on the regions to rename them as R7 and R4, respectively. Repeat the above steps to frame the 200 nm and 300 nm microspheres with rectangles successively and rename them as R5 and R6, respectively.
4. Finally, determine the 50-200 nm sorting region based on the electronic noise and the position of 200 nm particles defined as R8.
   NOTE: The lower detection limit of 50 nm is placed just above the electronic noise of the flow cell sorter. The region between the noise and the 200 nm microsphere population can therefore be defined to be between 50-200 nm.
5. Edit sort decisions in the sort logic and statistics panel. Double-click on the blank field of the left one (L1) stream. Select the region named R8 to create the sort logic. Choose the Abort mode of purity and select a droplet envelope of 1 drop for the L1 stream.
6. Load 500 µL sample of EVs mixture from step 2.3. Press the Start button under the acquisition menu in Summit to acquire data, and then press Start in the sort menu to collect 50-200 nm sEV into a 15 mL centrifuge tube.
   NOTE: A sterile environment is necessary to minimize contamination during any procedure.
7. Observe the presence of sEV by transmission electron microscopy (TEM) and verify the particle size of sEV using NTA. Perform western blot (WB) analysis to further confirm the expression of CD9, CD63, and CD81 markers¹⁸.