Anti cd63 antibody

Samples were first incubated
for 2 h with 3 mM PBASE (Sigma-Aldrich) in dimethylformamide (DMF)
(Sigma-Aldrich) at room temperature. Then DMF and DI water were used
to gently rinse the GFET sample to remove excessive PBASE from the
surface and dried with N₂. Then, the anti-CD63 antibody
(BD Bioscience US) were used. The antibodies were supplied in a stock
solution of 0.5 mg/mL in an aqueous buffer solution (containing ≤0.09%
sodium azide) and were diluted using 1 × PBS to a concentration
of 100 μg/mL. Droplets of 20 μL of 100 μg/mL anti-CD63
antibody were placed on the surface and left overnight in a humidified
environment at 4 °C. The samples were then sequentially rinsed
with 1 × PBS and DI water, and dried with N₂.

Exosomes isolated from TIG-3 cells were absorbed to formvar carbon-coated nickel grids and immune-labelled with an anti-CD63 antibody (BD, 556019), followed by 5 nm of a gold-labelled secondary antibody (British BioCell International Ltd.). The samples were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer, and a 2% phospho tungstic acid solution (pH 7.0) was used for negative staining. For the observation of cellular dsDNA, TIG-3 cells were plated on the gold disks and frozen in liquid propane at −175 °C. The samples were freeze substituted with 0.2% glutaraldehyde in acetone and 2% distilled water at −80 °C for 2 days. After dehydration, the samples were embedded into resin (LR White, London Resin Co. Ltd.) and ultra-thin sectioned at 80 nm using an ultramicrotome (Ultracut UCT, Leica). The samples were immunolabelled with an anti-dsDNA antibody (Santa Cruz, sc-58749) in normal goat serum and 1% BSA, followed by 10 nm gold-labelled secondary antibody. The grids were placed in 2% glutaraldehyde in 0.1 M phosphate buffer and dried. They were stained with 2% uranyl acetate for 15 min and a Lead stain solution (SIGMA). The samples were observed with a transmission electron microscope (JEM-1400Plus, JEOL Ltd.) at 80 kV. Digital images were obtained with a CCD camera (VELETA, Olympus Soft imaging solutions GmbH).

Exosomes isolated from cell culture medium were placed on nickel grids for 30 min at room temperature (RT) and were fixed in buffer (4% paraformaldehyde in 0.1 M Tris-HCl buffer [pH 7.4]) for 20 min at RT after removing the solution on the nickel grids. The nickel grids were then washed six times with 0.1 M Tris-HCl buffer (pH 7.4) and were blocked with 0.1 M glycine-Tris (pH 7.4) for 10 min at RT. After washing with 12 mM Na-P buffer, the nickel grids were blocked with 3% goat serum/0.1 M Tris-HCl buffer (pH 7.4) for 20 min at RT. The nickel grids were incubated overnight at 4 °C with anti-CD63 antibody (#556019, BD Biosciences, Franklin Lakes, NJ) diluted with buffer A (0.1 M Tris-HCl buffer [pH 7.4], 0.1 M PB) 1:20, followed by incubation with mouse anti-mouse IgG (10 nm gold colloidal particles, British Bio Cell International, Golden Gate, UK) diluted with 0.1 M Tris-HCl buffer (pH 7.4) 1:25 for 1 hour at RT. After washing with 0.1 M Tris-HCl buffer (pH 7.4) containing 0.1 M PB, the nickel grids were fixed with 2.5% GA in 0.1 M PB for 10 min at RT and were further washed with 0.1 M PB in Distilled Water. Finally, the grids were embedded in 1.5% uranyl acetate for 2 min at RT.

One milligram of aldehyde/sulfate latex beads (4 μm, Invitrogen) was incubated with 5 μg of purified anti-CD63 antibody (BD Pharmingen) under agitation overnight at 4°C. SF-derived EVs (1 × 10¹⁰) were incubated with 5 μg anti-CD63 beads in 100 μl PBS for 15 min at room temperature; the volume was made up to 300 μl and incubated for 2 hours at room temperature on a rotator. To block free binding sites, EV-coated beads were incubated for 30 minutes with 200 mM glycine. After two washes in PBS with 0.1% BSA, EV-coated beads were stained with the primary antibody anti-CD9 Alexa 647 (Serotec), anti-CD81 FITC (Biolegend), and anti-CD7 PE (Becton Dickinson) or isotype control (BD Biosciences) and analysed using a FACSCalibur flow cytometer (BD Biosciences).
Exosomes purified by immunoaffinity Exo-Flow kit were stained with specific monoclonal antibodies anti-CD81 FITC (Biolegend), anti-CD63 FITC (Santa Cruz), anti-CD9 PE (eBiosciences), anti-CD61 FITC (BD Biosciences), and anti-CD41a FITC (BD Biosciences) and analysed by flow cytometry.

EVs were incubated for 30 min with latex beads (Invitrogen). The EVs-coated beads were saturated for 30 min in PBS containing 5% BSA, then incubated for 1 h at 4 °C with an anti-CD63 antibody (556019; BD Biosciences). The beads were incubated with a biotin-coupled secondary antibody (E0433, Dako, Agilent Technologies Belgium, Heverlee, Belgium) for 30 min, incubated with streptavidin-PE (016-110-084, Jackson ImmunoResearch, Ely, UK) for 30 min, and analyzed on the FACSCalibur flow cytometer (BD Biosciences).

Flow cytometry was performed according to the method of Lässer et al. 30. Briefly, 4‐μm latex beads (Aldehyde/Sulfate Latex Beads 4 μm; Thermo Fisher Scientific) were coated with anti‐CD63 antibody (BD Biosciences, Oxford, UK). For each sample (antibody), EVs were added at a volume equal to a minimum of 15 μg of EV protein to ~100,000 beads. The beads and EVs were incubated at 4°C overnight under gentle movement. To block, the beads were incubated with 300 μl of 200 mM glycine for 30 min. The beads were washed (PBS containing 2% FCS) at 600 × g for 10 min., and this was repeated for a total of two washes. EVs were then stained against antibody of choice (CD63 eBioscience 12‐0639, CD9 eBioscience 1:20); after washing, the EV–bead complexes were resuspended in 500 μl PBS and assessed using a flow cytometer (Attune^® Acoustic Focusing Cytometer; Thermo Fisher Scientific).