Cd63 ab59479

Antibodies against EV biomarkers CD63, CD9 and Epcam were selected and prepared for capture and detectionfollowingthe manufacturer’s recommendations. By using commercialEV standards from the HCT116 cell line (HBM-HCT-30/5, Hansabiomed), different antibody combinations were compared and those with the best signals were selected. The antibodies used in the assayswere CD9 MA1-19,002 (ThermoFisher), CD63 ab59479 (Abcam) and Epcam MAB9601 (R&D Systems).
The preparation of SiMoa homebrew kits for EV detection followed the manufacturer’s guideline. In brief, the capture antibody concentration was adjusted to 0.2 mg/mL with Bead Conjugation Buffer (Quanterix) andparamagnetic carboxylated microparticles (Quanterix) were activated with 0.3 mg/mL 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (Thermo Fisher Scientific, Waltham, MA, USA). To start the biotinylation reaction, 3 μL of the biotin solution (2 mg NHS-PEG4-Biotin dissolved in 383 μL ddH₂O) were added to 100 μL of the detection antibody solution (1.0 mg/mL). The concentration of the recovered antibody was adjusted to 0.2 mg/mL and beads were stored at 4°C.
Finally, two different EVs detection assays were developed: the first assay detecting universal EVs via CD9-CD63 and the second assay detecting tumour-derived EVs via Epcam-CD63.

The total protein samples used for western blotting were obtained from lysates of differently treated cells using RIPA lysis buffer (Thermo Fisher Scientific, USA) and quantified using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Equal amounts (25 μg) of protein samples were loaded onto a 10% acrylamide gel for SDS-PAGE according to molecular weight. Proteins from the gel were transferred to a nitrocellulose membrane (Millipore, USA) through electrophoresis. The membrane was blocked for 1 h with 5% nonfat dry milk. Overnight incubation with specific antibodies (NCOA4, bs-19051R, Bioss, USA; GAPDH, #5174, CST; TSG101, Ab125011, Abcam, UK; CD63, Ab59479, Abcam, UK) at 4 °C and 1 h incubation with a secondary antibody (Beyotime, China) at 37 °C were performed in the same order. The blot on the membrane was visualized using a chemiluminescence system (Tanon, China). GAPDH expression level was used as the control.