Exosome depleted fcs

EVs were isolated and characterized according to the 2018 consensus statement on minimal information for studies of extracellular vesicles (MISEV2018) (6 (link)). Cells were cultured for 48 h in DMEM supplemented with 10% exosome depleted FCS (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and 1% penicillin-streptomycin. Supernatants (30 ml) were collected and centrifuged for 10 min at 300 g to remove cells and cell debris, followed by 30 min at 10000 g to remove larger vesicles. Afterwards the supernatants were filtered through a 0.2 µm filter and centrifuged at 100000 g for 1.5 h. Pelleted EVs were washed with PBS and centrifuged for another 1.5 h at 100000 g. Centrifugation was performed using a Sorvall WX+ Ultra Centrifuge, with SureSpin 632 rotor (k-factor 194) (Thermo Fisher Scientific, Waltham, Massachusetts, USA). EVs were resuspended in PBS. EV analysis was performed by nanoparticle tracking analysis (NTA) using a NanoSight NS300 (Malvern Panalytical, Kassel, Germany). Therefore, EVs were isolated or samples were diluted (conditioned media (1:100) or patient serum (1:1000)) without isolation and analysed from three independent biological samples. Measurements were performed at a controlled temperature of 22°C. For each sample, three measurements of 30 s were performed. EV concentration and size was calculated by the NanoSight software.

Cells were cultured for 48 h or 72 h in DMEM supplemented with 10% exosome-depleted FCS (Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin. Supernatant was collected and centrifuged for 5 min at 1000 rpm to remove cells and cell debris, followed by 30 min at 6000 rpm to remove larger vesicles. Afterwards, the supernatant was filtered through a 0.2 µm filter and centrifuged at 100,000× g for 2 h. Pelleted exosomes were washed with PBS and centrifuged for another 2 h at 100,000× g. Exosomes were resuspended in PBS. Alternatively, exosomes were isolated using an exosome isolation reagent for cell culture (Thermo Fisher Scientific), following the manufacturer’s instructions. Exosome analysis was performed by nanoparticle tracking analysis (NTA) using a NanoSight NS300 (Malvern Panalytical, Kassel, Germany). Isolated exosomes were stained with SYTO^® RNASelect™ (Thermo Fisher Scientific) regarding the manufacturer’s instructions and analyzed by fluorescence microscopy and flow cytometry.