Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

The institutional ethical board of the University of Dusseldorf has approved the experiments presented in this work (Reference number: 3381).

1. EV Isolation from Human Whole Blood

1. Isolate EVs with exosome precipitation solution.
   1. Collect 2 mL of human whole blood in serum-separating tubes (SST) via venipuncture and incubate the tube for 15 min at room temperature (RT) until coagulation is finished.
   2. Centrifuge the SST at 1,700 x g for 10 min at RT to separate cells from serum and transfer 1 mL of the serum to a 1.5 mL reaction tube. Centrifuge the platelet-rich plasma (PRP) at 3,000 x g for 15 min at 4 °C to remove platelets and transfer 100 μL of the platelet-poor plasma (PPP) to a new 1.5 mL reaction tube.
   3. Add 25 µL of exosome precipitation solution (4 parts PPP, 1 part exosome precipitation solution) and vortex thoroughly. Incubate the sample for 30 min on ice. Keep the tube upright and do not rotate or mix the tube during the incubation period.
   4. Centrifuge the sample at 1,500 x g for 30 min at 4 °C to pellet the EVs. After centrifugation, the EVs appear as a beige or white pellet at the bottom of the vessel. Aspirate the supernatant and centrifuge the sample at 1,500 x g for 5 min at 4 °C.
   5. Remove all traces of fluid and re-suspend the pellet in 100 µL of phosphate-buffered saline (PBS) by frequently pipetting up and down. Store the EV suspension at -80 °C when analysis is not performed immediately.
      Note: When isolating EVs from plasma, fibrinogen and fibrin can impede efficient recovery and resuspension is heavier and takes more time.
2. Isolate EVs with ultracentrifugation.
   1. Take the pre-cooled rotor (TLA-55 fixed-angled) from the fridge and cool down the ultracentrifuge before use.
   2. Transfer 1.25 mL of PPP (prepared in step 1.1) to a suitable 1.5 mL ultracentrifugation tube with cap and centrifuge the sample at 110,000 x g for 90 min at 4 °C. Make sure that the rotor load is balanced before starting.
   3. Decant the supernatant and place tube upside-down on a paper towel for 2 min. Re-suspend the pellet in 500 μL of PBS and centrifuge the sample at 110,000 x g for 90 min at 4 °C.
   4. Aspirate the supernatant and re-suspend the pellet in 50 μL of PBS.
      Note: Use only rotors and accessories designed for the ultracentrifuge that is in use. Pretest the tubes in the rotor by using water because the strength of the tubes can vary between lots.

2. Staining of the Samples

1. Stain samples with PKH67.
   1. Prepare the staining solution by adding 1 μL of the PKH67 ethanolic dye solution to 50 μL of diluent C (included in the kit) in a 1.5 mL reaction tube and mix thoroughly.
   2. Transfer 10 μL of the staining solution to 20 μL of the EV suspension (prepared in step 1.1) to the reaction tube, mix thoroughly and incubate for 5 min at RT in the dark.
   3. Dilute 50 μL of the stained EV suspension with 2.5 mL distilled water in a 15 mL reaction tube, mix thoroughly, and use this final suspension for particle measurement.
      Note: It is crucial to use water as diluent for the EV suspension, because other diluents (e.g., PBS) can impair the measurement. When using stored EV-suspension, thaw the samples on ice and mix thoroughly before staining.
2. Stain samples with specific antibodies.
   1. Dilute 10-20 μL of the EV suspension (prepared in step 1.1) with 50 μL of distilled water in a 1.5 mL reaction tube and mix thoroughly.
   2. Add 2.5-5 μL of the specific antibody (Table 1) to the reaction tube, mix thoroughly, and incubate for 30 min at RT in the dark.
   3. Transfer 50 μL of the stained EV suspension to 2.5-10 mL of distilled water (Table 1) in a 15 mL reaction tube, mix thoroughly, and use this final suspension for particle measurement.
      Note: Under some circumstances, the concentration of the used antibody must be adjusted (Table 1).

3. Processing of the Samples

Note: The fundamentals of this method were extensively described previously and below the protocol focuses on the specific steps needed for analysis of fluorescence-labeled EVs¹⁴.

1. Perform the startup procedure.
   1. Push the fluorescence filter into the optical path of the microscope and camera. Start the program and follow the instructions on the screen for automated implementation.
   2. Select the correct cell number (Z158_C1149_Fluor) in the “Cell Check” tab (cell definition) for fluorescence measurement. Select the reference position for the optics to make sure that laser and microscope are in a common focus (laser and microscope move to this position automatically).
   3. Flush the cell channel with a syringe filled with 10 mL of distilled water. Ensure that the measurement cell is free of air bubbles and do not inject air bubbles into the system.
   4. Prepare a calibration suspension containing uniform 200 nm sized fluorescence-labelled polystyrene particles that have carboxylate groups on their surface. Dilute 10 μL of the particles with 990 μL distilled water. Then dilute 10 μL of this particle solution in a 15 mL tube with 10 mL of distilled water to obtain the required concentration.
   5. Inject 2.5 mL of the diluted particle solution in the cell channel and click “Optimize focus” to adjust the camera.
2. Measure the sample.
   1. Flush the cell channel multiple times with a syringe filled with 10 mL of distilled water prior to each sample measurement. Inject the stained EV suspension (prepared in step 2) into the cell channel.
   2. Adjust the following main camera parameters in the “Cell Check” tab in the software as needed (Table 2). Use the reference position or position 0.41193 to adjust the parameters.
      1. For sensitivity, find the optimal sensitivity range by clicking on the button “Number of Particles vs. Sensitivity” to display a curve of measured particles per screen for different sensitivity levels.
         Note:
      2. For shutter, adjust the period of time that the camera allows light to pass for a determined interval.
      3. For post-acquisition parameters, choose a minimum brightness of 20, a minimum size of 20 nm, and a maximum size of 500 nm for measurement.
   3. Note the number of detected particles counted in the field of vision from the display. The scattering bar must be in the green to orange range (50-300 particles). If the scattering bar is red, the scatter will fuse individual particles and they are counted as one single particle, thus leading to false results. In such a case, further dilute the sample to avoid overlapping of the particles or lower the sensitivity.
   4. Click on “Check particle drift at 0 V” in the “Cell Check” tab before starting the measurement. If the drift is higher than 5 μm/s, wait until the sample stops flowing to continue the measurement.
      Note: If the drift is too high at the beginning of the measurement, the repeated measurements can differ and sophisticate the results. Due to the underlying principles of NTA, a relevant drift may have an impact in the determined particle size, as calculated by the software.
   5. Click on “Run Video Acquisition” in the “Measurement” tab. Select the number of experiments (3-5) and the time delay between them (0 min).
   6. Define the number of (individual subvolume-) positions (11) and the number of (measurement) cycles (10) at each measurement position, where particles should be analyzed
   7. Select a folder, create a new file name, and click “OK” to start the measurement.

4. Interpretation of the Results

1. View the results and parameters on the “Analysis” tab after the measurement. Check the following parameters after the analysis before cleaning the cell channel: average number of particles per position, total number of traced particles and particle concentration, the distribution width of the particles (x10, x50, and x90 values), value of mean and standard deviation. Repeat the measurement of the sample if required.
   Note: The results are also saved as a .pdf or .txt file.
2. Click on “Analysis” tab to view the graph calculated after the measurement, which shows the distribution of the detected particles by size. Click on “Display” in the “Analysis” tab and use the icons to adjust and change the graph settings for particular requirements.

5. Validation via EV Detection by Western Blotting

1. Dissolve the EV pellet (prepared in step 1) in RIPA lysis and extraction buffer, pipette thoroughly and incubate on ice for 30 min. Centrifuge the sample at 8,000 x g for 10 min at 4 °C to clarify the lysate and transfer the supernatant to a new tube.
2. Measure the total protein by a Lowry protein assay kit. Dilute 1 μL of the isolated EV suspension with 49 μL of RIPA buffer and use 5 μL in triplicates for analysis. Dilute the EV suspension with 2x Laemmli loading buffer to a final concentration of 2 µg/μL and heat for 10 min at 95 °C.
3. Load 20 μg of protein per well. Separate and transfer the proteins by polyacrylamide gel electrophoresis and tank blotting according to standard protocols.
4. Block the membrane (0.2 μm polyvinylidene difluoride) with bovine milk powder (5%) for 1 h at RT and incubate the membrane with specific primary antibodies (CD9, CD63, and vimentin) overnight at 4 °C.
   Note: The primary antibodies are used at a 1:1,000 dilution.
5. Wash the membrane with TBST 3x 5 min and incubate the membrane with horseradish peroxidase (HRP)-conjugated secondary antibody (1:20,000 in TBST) for 60 min at RT.
6. Wash the membrane with TBST 3x 5 min and detect the proteins by chemiluminescence with a high sensitivity substrate solution on an imaging system.
   Note: Because CD63 antigen is extensively and variably glycosylated, the molecular weight can vary, and bands can appear between 40-65 kDa.