Anti cd9

Primary myeloma cells, myeloma cell lines and breast cancer cell line were used for exosome production. In brief, cells were cultured in respective media with microvesicle-free fetal bovine serum for 48 hours. Conditioned media was collected, centrifuged twice at 3000 rpm for 10 min to remove debris. The supernatant was centrifuged at 100000 × g for 60 min to collect exosomes.
The pellet containing exosomes was resuspended in 1 × PBS buffer. They were examined by transmission electron microscopy (High Resolution Electron Microscopy Facility at Xiamen University). The hydrodynamic size distribution of exosomes were determined by dynamic light scattering (DLS) system. Purified exosomes were incubated with 4 μm-diameter aldehyde/sulphate latex beads (Interfacial Dynamics) in PBS buffer overnight at 4°C. Exosome could be stained with exosome marker antibodies, anti-CD9 (BioLegend) or anti-CD63 (BioLegend) for 30 min at 4°C, and analyzed using flow cytometer (BD Biosciences).

The ELISA for exosome-associated TGFβ1 was done using a modification of the methods of Logozzi et al^{10 (link)} and Sullivan et al.^{1 (link)} Half-area, high binding microtiter plates were coated with capture antibodies to LAP-TGF-β1 (BioLegend, Clone TW7-16B4); LTBP I, II, or III (Bioss Antibodies); GARP (Biolegend F011-5); or TGFβ1 (Abcam) at 10 µg/mL each in 10 mmol/L TRIS pH9 and incubated overnight at 4 °C. After blocking plate with 2% BSA in PBS for 3 h at room temperature, tested samples were added, and plates were incubated overnight at 4 °C. Wells were washed, in PBS/0.1%BSA, and bound target antigen was detected with the following biotinylated anti-tetraspanin monoclonal antibodies: anti-CD81 (Thermo Fisher, MA5-1793), anti-CD9 (Biolegend, MZ3), or anti-CD63 (Biolegend, NVG-2) at 1 µg/mL concentration. After 3 h of incubation at room temperature, wells were washed, and HRP-Streptavidin (Biolegend) was added at 1:1000 dilution. After 45 min incubation, wells were washed, and a solution containing 3,3′,5,5′-tetramethylbenzidine (TMB) was added. The reaction was stopped after 10 min with TMB stop solution, and color development was read at 450 nm.

For analyzing expression of EV biomarkers and EV quantification the ExoView R100 platform (NanoView Biosciences, Boston) was used. Human ExoView Tetraspanin (EV-TETRA-C) kit was used for the analysis. The samples (EV, total sweat and patch) were processed according to the manufacturer's protocol. 1 µg of protein samples were carefully loaded onto each chip and incubated for 24 h. After that, the chips were washed three times on an orbital shaker to remove unbound particles. The chips were incubated for one hour with the human anti-CD81 (BD Pharmingen 555675), anti-CD63 (BD Pharmingen 556019), and anti-CD9 (Biolegend V P018) fluorescently labelled antibodies. Mouse IgG (Biolegend 400101) were used as controls. The immunostained chips were washed three times in PBS, once in deionized water and dried. Imaging and data acquisition of the stained chips were performed with the ExoView R100 (NanoView Biosciences) and the data analysis with the ExoViewer 3 (NanoView Biosciences).

To analyze cell surface maker expression, the cells were incubated with specific antibodies for 30 min at room temperature. The following antibodies were used: anti‐CD45 (eBioscience, 30‐F11), anti‐CD11b (Biolegend, M1/70), anti‐Ly6G (eBioscience, RB6‐8C5), anti‐CD9 (Biolegend, MZ3) and anti‐CD47 (Biolegend, miap301).