4.4. BCA and Exosome Concentration
To determine the protein concentration of the isolated exosome fraction, Pierce BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA, 23225) was applied according to manufacturer’s protocol. Exosomes were concentrated using 100 kDa cutoff centrifugal filter units (Millipore, Burlington, MA, USA, UFC5100BK). For on-bead flow-cytometry, 10 µg of exosomes in 100 µL PBS were used, for Western blot analysis, 20 µg of exosomes in 40 µL PBS were used.
4.5. Characterization of Exosomes
Freshly isolated exosomes were prepared for TEM by negative staining. Therefore, 5 µL of the exosome solution was added onto a glow discharged carbon-coated copper TEM grid and incubated for 1 min. Then, the grids were washed 3 times with a droplet of water. Next, the grids were incubated three times with droplets of 3% uranyl acetate in water for 30 s each. Afterward, the grids were dried on air with a small amount of uranyl acetate left. The grids were imaged with a JEOL 1400 TEM (Freising, Germany).
NTA was performed using ZetaView PMX-220 (Particle Metrix, Inning am Ammersee, Germany). Freshly isolated exosomes were diluted 1:100,000 in 1 mL PBS and loaded into the cell. Measurements were performed at 11 different positions throughout the cell, with three cycles of records at each position. Instrument parameters were set to a temperature of 25 °C, a sensitivity of 85 and a shutter of 100. PBS and 100 nm polystyrene beads were used as controls. For data recording and for calculating nanoparticle size ranges and concentrations, corresponding ZetaView 8.05.11 software was used.
For Western blots, 20 µg exosomes/lane were mixed at a v/v ratio of 1:5 with Pierce Lane Marker Reducing Sample Buffer (ThermoFisher Scientific, Waltham, MA, USA, 39000) for detection of CD9 and TSG101 or with Pierce Lane Marker Non-Reducing Sample Buffer (ThermoFisher Scientific, Waltham, MA, USA, 39001) for detection of CD63 and CD81 and denatured for 5 min at 95 °C. Samples were separated on 12% Mini-Protean TGX Precast Gels (BioRad, Hercules, CA, USA, 4561044) and transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer System (BioRad, Hercules, CA, USA). Membranes were incubated overnight at 4 °C with the following primary antibodies: anti-TSG101 (Invitrogen, Carlsbad, CA, USA, PA5-31260, 1:500), anti-CD9 (Invitrogen, Carlsbad, CA, USA, 10626D, 1:1000), anti-CD63 (Invitrogen, Carlsbad, CA, USA, 10628D, 1:1000) and anti-CD81 (Invitrogen, Carlsbad, CA, USA, 10630D, 1:1000). Horseradish peroxidase (HRP)-conjugated secondary antibodies (ThermoFisher Scientific, Waltham, MA, USA, 31450 and 31460, 1:10,000) were added for 40 min at RT. Bands were detected using SuperSignal West Dura Extended Duration Substrate (ThermoFisher Scientific, Waltham, MA, USA, 34076) and ChemiDoc XRS+ Imaging System (BioRad, Hercules, CA, USA).
These methods for exosome characterization are in line with the MISEV 2018 guidelines for the definition of extracellular vesicles [29
] and are routinely performed as described in detail in our previous publications [16
4.6. Flow Cytometry of Cells
Cultured cells were detached from flasks using TrypLE Express Enzyme (Gibco, Carlsbad, CA, USA, 12605-010) and washed twice with FACS buffer (PBS supplemented with 2% bovine serum albumin (BSA)). 106 cells were stained in 100 µL FACS buffer using 5 µL of PE-Cy7 conjugated anti-CD16 (BD, Franklin Lakes, NJ, USA, 335788) for 30 min at 4 °C in the dark. After two further washing steps, cells were measured in a Gallios flow cytometer using Kaluza 1.0 (Beckman Coulter, Brea, CA, USA) and data were analyzed using Kaluza Analysis 2.1 software.
4.7. Immune Capture and On-Bead Flow Cytometry of Exosomes
Exosomes were first captured on ExoCap Streptavidin magnetic beads (MBL Life Science, Woburn, MA, USA, MEX-SA) as previously described [18
]. Briefly, exosomes (10 µg in 100 µL PBS) were incubated for 2 h at RT with biotin-labeled anti-CD63 (BioLegend, San Diego, CA, USA, 353018) for total exosome capture or with biotin-labeled anti-CD44v3 (R&D Systems, Minneapolis, MN, USA, BBA11) for the enrichment of TEX. Biotin-labeled anti-CD63 was adjusted to a concentration of 1 µg in 100 µL PBS. Anti-CD44v3 was custom biotinylated using Lightning-Link Rapid Biotin Antibody Labeling Kit (Novus Biologicals, Littleton, CO, USA, 370-0010) and used at 2 µg in 100 µL PBS. Next, an aliquot of beads (10 µL for CD63, 100 µL for CD44v3) was added and samples were again incubated for 2 h at RT. The uncaptured fraction was removed and samples were washed using a magnetic rack.
For CD16 detection by on-bead flow cytometry, the bead/anti-CD63 or anti-CD44v3/exosome complexes were incubated with 5 µL of PE-Cy7 conjugated anti-CD16 for 1 h at RT. An appropriate isotype control (BD, Franklin Lakes, NJ, USA, 348788) was used. Next, the stained complexes were washed three times with PBS using a magnetic rack and were finally resuspended in 300 µl PBS for flow cytometry. Detection was performed using a Gallios flow cytometer with Kaluza 1.0 software (Beckman Coulter, Brea, CA, USA). Samples were run for 2 min and around 10,000 events were acquired. Gates were set in the bead fraction visible in the forward/sideward light scatter. Data are presented as RFI which equals the mean fluorescence intensity (MFI) of the stained sample divided by the MFI of the isotype control.