Exoquick ultra ev isolation kit for serum

RCC cells were cultured in medium with 10% exosome-free medium (System Biosciences) for 72 h before the isolation of exosomes. The supernatant was collected and centrifuged at 300 × g for 10 min, 2000 × g for 10 min, 10,000 × g for 30 min, in turn. After filtration with a 0.22-pm filter (Millipore, USA), the filtrate was centrifuged at 120,000 × g for 70 min twice (Beckman Coulter, USA). The pellets were resuspended with PBS and prepared for the subsequent experiments. For transmission electron microscopy, exosomes were fixed with 2% paraformaldehyde and placed on 200-mesh Formvar-coated grids. The grids were then stained using 2% phosphotungstic acid for 2 min and observed on a transmission electron microscope (Hitachi H-7500). The size of exosomes was detected by Nanosight ns300 (Malvern Instruments Ltd., UK). For exosomes labeling, exosomes were labeled using PKH67 membrane dye (Sigma). Labeled exosomes were collected by ultracentrifugation after washing in 10 mL PBS, and resuspended in PBS. For cell treatment, exosomes were incubated with recipient cells for 48 h. An ExoQuick ULTRA EV Isolation Kit for Serum (System Biosciences) was used to isolate the exosomes from the serum of RCC patients. The RNAs in exosomes were extracted using an Exosome RNA Purification Kit (EZBioscience, USA).

A total of 250,000 CA1a or MB‐231 cells were resuspended in PBS‐Matrigel (Corning, USA) solution (1:1 ratio) and implanted in the flanks of 8‐week‐old female NSG mice. When tumours reached 15 mm in diameter, the mice were sacrificed, and the whole blood was taken collected. The blood was allowed to clot undisturbed for 30 min at RT and then centrifuged at 1500 × g for 15 min at 4°C. We collected the serum from the resulting supernatant and pooled the serum from two mice of the same group. Circulating EVs were prepared from equal volumes of serum inputs using ExoQuick Ultra EV Isolation Kit for Serum (System Biosciences, USA), following the manufacturer's protocol. We then incubated the isolated circulating EVs with anti‐human CD63 magnetic beads (Thermo Fisher, USA), and stained them with fluorophore‐conjugated anti‐human ITGAV antibody (BioLegend, USA) for flow cytometry analysis, as described above.