Surespin 632 rotor

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.

Lentiviruses were produced as previously described.⁶⁷ (link)^,68 In brief, HEK293T cells were transfected with the shRNA-expressing pLKO plasmids, together with two packaging plasmids, psPAX2 and pMD2.G, using PEI MAX (Polysciences, Warrington, PA, no. 49553-93-7). The supernatant was collected at 3 days post-transfection and concentrated with a 20% sucrose cushion by ultracentrifugation in a SureSpin 632 rotor (Thermo Scientific) at 19,400 rpm for 3 h. The transduction unit of the produced lentiviruses were titrated as described previously.⁶⁹ (link) Proliferating CuFi-8 cells were transduced at an MOI of ∼5 transduction units/cell. At 3 dpt, the cells were treated with puromycin at a final concentration of 2.5 μg/mL to select the transduced cells.