Immunocapture and Flow Cytometry of Extracellular Vesicles for Antigenic Characterization

1. Coupling of Magnetic Nanoparticles (MNPs)

1. Use commercial coupling procedure and reagents to couple MNPs with antibodies (Abs) (typically 1 mg). Iron oxide nanoparticles with carboxylic acid as reactive groups are used.
   1. If antibodies are in a volume higher than 0.5 ml, concentrate using a 100K concentrator. Spin at 2,000 x g for 3-5 min.
2. At the end of the procedure, after adding 10 µl of the quenching buffer, transfer MNPs to a 12 mm x 75 mm tube and add 3 ml wash/storage buffer. Place the tube in the center hole of a magnetic separator and leave overnight at 4 °C.
3. Verify that MNPs have all collected to the side of the tube, and then carefully pipet out all liquid. Add 3 ml of fresh wash/storage buffer, and place back in the magnet.
4. After several hr, check if MNPs are collected to the side of the tube and then pipet off the liquid. Use 2 ml of wash/storage buffer to resuspend the MNPs and store at 4 °C.
5. Verify that Abs are coupled to MNPs by labeling them with a relevant fluorescent Fab antibody fragment (e.g. goat anti-mouse if using a mouse monoclonal Ab for capture as described in section 2) and run on a flow cytometer.

2. Labeling Antibodies Coupled to MNPs

1. Combine 60 µl (3.9 x 10¹² particles) of MNPs (per condition) and 5 µl (of a 200 µg/ml commercial solution) labeled Fab fragments, specific for the isotype of the capture Ab in a 1.5 ml microcentrifuge tube.
2. Incubate at room temperature (RT) for 30 min with continuous mixing.
3. Pre-wet a 100K concentrator with 300 µl of phosphate buffered saline (PBS) and spin in a microcentrifuge at 1,500 x g for 5 min.
4. Purify labeled complexes on 100K column. Spin the mixture from step 2.2 in a microcentrifuge at 1,500 x g for 5 min, wash with 200 µl PBS, and recover in the initial volume.

3. Use of Labeled Ab-MNP Complexes to Capture Particles of Interest (Extracellular vesicles, EVs)

1. Incubate pre-labeled MNPs 60 µl (3.9 x 10¹² particles) with EV preparation (100 µl).
   1. Prepare EV preparations using various methods. Here, purify EVs on sucrose gradients, collect them from platelet-poor plasma using exosome precipitation reagents or isolate directly from plasma.       
      NOTE: Optimal ratios need to be determined for each experiment and are dependent on the concentration of EV in the preparation. MNP-Ab fraction should be ~10⁶ in excess compared to concentration of EV fraction.
2. Incubate 1 hr at 4 °C for EVs.
3. Add 2.5% mouse IgG/ human IgG to block Fc binding, gently vortex, incubate 3-5 min at RT.
4. Add manufacturer recommended or titered concentrations of each detection Abs and incubate additional 20 min at RT.

4. Separation of the MNP-captured EVs from Unbound Antibodies Using Magnetic Columns

1. Prepare magnetic separation column for use by placing it in a separator magnet.
2. Pre-wet the column with 500 µl of wash buffer (2 mM Ethylenediaminetetraacetic acid (EDTA), 0.5% bovine serum albumin (BSA) in PBS). Allow the wash buffer to flow through the column.
3. Add the MNP-EV complexes to the column and allow all liquid to flow through. Add 500 µl of wash buffer to the column, allowing the whole volume to pass through.
4. Repeat washing with wash buffer 2 more times.
5. Remove the column from magnet and place in 12 mm x 75 mm tube for collection of the retained MNP-EV complexes. Let the column stand in the tube off the magnet for 3 min. Add 200 µl of PBS and let the beads flow down by gravity, add another 200 µl of PBS, and fix with 200 µl 4% paraformaldehyde after elution.
   NOTE: Paraformaldehyde is a suspected carcinogen and should be handled in a ventilated hood and gloves should be worn.
6. To quantify single EVs use volumetric settings on High Throughput Sampler (HTS) or just prior to acquisition add well-mixed flow cytometry count beads.

5. Analysis of MNPs-captured EVs with a Flow Cytometer

1. Warm up flow cytometer for 30 min.
2. Run quality control beads.
3. Set threshold on fluorescence of either MNPs or labeled EVs. Use 0.22 µm filtered PBS for threshold to decrease the background "noise". Set the threshold by adjusting the PMT voltage at the level where filtered PBS gives no, or very few, events.
4. Run samples on low. (Use an additional 0.04 µm inline filter for sheath fluid placed in series behind the standard 0.2 µm sheath filter to further eliminate false events).