Aldehyde sulphate latex beads

Aliquots of exosomes (about 30 μg vesicular protein) were incubated with 3 × 10⁵ 4 μm-diameter aldehyde/sulphate latex beads (Thermo Fisher Scientific, USA) labeled anti-CD9 antibody (ab134375, Abcam) in 150 μL of PBS at 4 °C overnight at gentle agitation and blocked in 0.2 M glycine for 30 min. The exosomes-antibody-bead complexes were washed twice with a washing buffer (2% EVs depleted bovine serum in PBS) and stained with anti-Tspan8-PE antibody (3 μL on test, ABIN4895321, Antibodies-online, Germany) and anti-CD151-APC antibody (3 μL on test, #350405, Biolegend, San-Diego, CA, USA) for 20 min at room temperature. All complexes were washed twice, suspended in 300 µL of FACS buffer and analyzed by flow cytometry on cytometer Cytoflex (Becman Coulter, Malaysia). Data were analyzed with CytExpert 2 Software. Single beads were gated, and tetraspanin compositions (%) at the surface of exosomes were calculated.

Exosomes were enriched and depleted from conditioned medium by ultracentrifugation. Medium was centrifuged at 4 °C for 30 min at 2,500×g (Thermo Fisher, Heraeus Multifuge 3SR+). The supernatant was centrifuged at 4 °C for 35 min at 4,565×g, passed through a 0.2 μm filter, and centrifuged at 4 °C for 2 h at 110,000×g in an Optima XPN ultracentrifuge with a SW32 Ti rotor (Beckman Coulter). The cleared supernatant was the exosome-depleted fraction; pellets were resuspended in Cor.4U medium as the exosome-enriched fraction. For exosome identification, 75,000 4 μm aldehyde/sulphate latex beads (Thermo Fisher) were resuspended in 0.025 mol/l MES buffer [2-(N-Morpholino)ethanesulfonic acid; Sigma-Aldrich], coated with 8 μg CD63 (Biolegend) or rat IgG2α (Biolegend), incubated on a rotator for 20 min in a final volume of 50 μl PBS, then incubated for 30 min with 100 mmol/l glycine (Sigma-Aldrich) to occupy unreacted sites. Media were incubated with the antibody-coated beads in a final volume of 250 μl for 15 min. Exosome-bead complexes were stained with 10 μg/ml FITC labelled anti-CD9 (Biolegend), with gentle agitation for 30 min in the dark. Results were visualised using an LSRII flow cytometer (Becton Dickinson) equipped with five lasers^{5 (link)}, and were analysed using FlowJo (v10, FlowJo).

EVs (30 µg) were incubated with 1 µl aldehyde/sulphate latex beads (Invitrogen) (total volume, 500 µl PBS) for 15 min (room temperature) with continuous rotation. The reaction was stopped using 100 mM glycine and 2% BSA in PBS for 30 min (room temperature) with continuous rotation. Beads were centrifuged at 5000 × g (2 min), washed again with 300 µl PBS, permeabilized (0.2% TX-100 in PBS for 5 min), blocked (0.2% TX-100, 10% BSA in PBS for 10 min), washed and incubated with anti-MKLP1 antibody (1:50 dilution in 0.2% TX-100/2% BSA in PBS for 1 h) at room temperature with continuous rotation. Beads were washed 3× and incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen) at 1:100 dilution in 0.2% TX-100/2% BSA in PBS for 20 min under continuous rotation. Beads were washed 3×, resuspended in PBS and imaged using Zeiss AxioObserver Z1 microscope (Zeiss).

Fractions from SEC and EV-enriched fractions from PEG and PROSPR were analyzed by flow cytometry to identify the classical exosomal markers CD63, CD9 and CD81. In the same way as previously reported14 , 50 μL of each fraction were incubated with 0.2 μL aldehyde/sulphate-latex beads (4 μm; Invitrogen, Carlsbad, CA) for 15 min at RT. Once re-suspended in 1 mL bead-coupling buffer (BCB) (PBS supplemented with 0.1% BSA and 0.01% NaN3; Sigma Aldrich), the mix was incubated overnight at RT on rotation. EV-coated beads were then spun down at 2,000× g for 10 min, washed with BCB and centrifuged again at 2,000× g for 10 min. EV-coated beads were then labelled at 4 °C with anti-CD9 (Clone VJ1/20), anti-CD63 (Clone TEA 3/18), and anti-CD81 (clone #G0709, from Santa Cruz Biotech) or polyclonal IgG isotype (Abcam, Cambridge, UK) antibodies for 30 min. After washing with BCB, EV-coated beads were incubated with FITC-conjugated secondary goat anti-mouse antibodies (SouthernBiotech, Birmingham, AL) for 30 min, washed twice with BCB and analyzed by flow cytometry (FacsVerse; BD Biosciences, San Jose, CA) using the FlowJo software (Tree Star, Ashland, OR). A total of 10,000 beads were acquired for each sample and the median fluorescence intensity (MFI) was obtained.