Minute hi efficiency exosome precipitation reagent

EVs were collected by density gradient ultracentrifugation as described previously.^[62] THP‐M culture medium was centrifuged at 800 × g for 5 min, followed by centrifugation at 2000 × g for 10 min to eliminate cellular debris. The supernatant was then passed through a 0.22‐µm PVDF filter (Millipore, USA) and centrifuged at 100 000 × g for 90 min at 4°C. The supernatant was discarded, and the pellets were washed with PBS and used to suspend the pellets. EVs were isolated from plasma of patients with NSCLC by use of Minute Hi‐Efficiency Exosome Precipitation Reagent (Invent Biotechnologies, USA). For removing large debris, 1‐mL plasma samples were centrifuged for 10 min at 2000 × g. Two volumes of EVs precipitation reagent were added to the supernatant, followed by 1 h of incubation. Purified EVs were obtained by centrifugation of the sample for 30 min at 10 000 × g, followed by centrifugation at 100 000 × g for 70 min. The size distribution and concentration of EVs were determined with a ZetaView particle tracker from ParticleMetrix (Germany). The shape of EVs was assessed with a transmission electron microscope (Tokyo, Japan). CD63, Alix, and TSG101 served as markers of EVs; GM130 served as a marker of cells. The concentrations of EVs were quantified with BCA protein assay kit (Beyotime Biotechnology, China).

Exosomes were isolated by Minute™ Hi-Efficiency Exosome Precipitation Reagent (Invent EI-027) according to the protocol and then filtered with a 0.22 μm filter to purify the exosomes. The exosomes were identified by transmission electron microscope observation and exosomal protein biomarkers. The exosomes were fixed with 4% glutaraldehyde at 4° C for 2h, and then rinsed three times with 0.1mol/L PBS and fixed with 1% osmium tetroxide for 2h. Next, dehydrated by conventional ethanol and gradient acetone, exosomes were immersed, embedded, polymerized in epoxy resin and then observed under a transmission electron microscope. The proteins from exosomes were lysed with cell lysis buffer (Takara 635656). CD63 (Abcam 125011) and TSG101 (Abcam 134045) were used as exosome markers.