Mla 55

For sEV isolation, cells were incubated for 72 h. Then, medium was collected and the cells and cellular debris were removed from the medium by subsequent centrifugation at 300× g for 10 min and 2000× g for 10 min, respectively. The medium was passed through a 0.22 μm-pore-size filter (cellulose acetate membrane, Corning) to remove larger vesicles. The sample was concentrated (Amicon^® Ultra, with Ultracel-100 regenerated cellulose membrane; Merck, St. Louis, MO, USA) and ultracentrifuged on 2 mL of 20% sucrose cushion for 135 min, 4 °C at 100,000× g (MLA-55; Beckman Coulter) [72 (link)]. The pellet was resuspended in PBS and subsequently used for nanoparticle-tracking analysis (NTA), transmission electron microscopy, in radioimmunoprecipitation assay buffer (RIPA) for Western blot, and in lysis buffer (mirVana; ThermoFisher) for RNA extraction studies.

One mL of plasma was diluted with 8.5 mL particle-free Dulbecco’s Phosphate buffer saline (dPBS, Sigma-Aldrich, USA), carefully overlaid on 2 mL of 20% sucrose cushion (sucrose (Merck Millipore, USA) in dPBS) in polypropylene tubes (Beckman Coulter, USA) and centrifuged at 100,000×g for 135 min at 4 °C (MLA-55, Beckman Coulter, USA) to enrich EVs in the sample. The supernatant was gently aspirated and residual moisture absorbed from the walls of the tubes. The pellet was resuspended in small volume of dPBS (NTA: 20 µL, AF4-UV-MALS: 40 µL, TEM: 20 µL) and stored at − 20 °C. For miRNA extraction, pellet was resuspended in 200 µL of dPBS and 800 µL Tri-reagent (Sigma, USA) was added before storage.
To analyze repeatability of sUC EV-enrichment method linked to quantification methods, 4 aliquots of plasma from the same donor were processed as described above. Repeatability is measured by % relative standard deviation (%RSD) and is calculated as: (standard deviation/mean)*100.

Each 10⁶ HEK293T cells were grown in DMEM with 10% FCS and each 1% Penicillin and Streptomycin (Life Technologies, Darmstadt, Germany) at 37 °C, 5% CO₂. On the day of transfection, fresh DMEM with 25 μM chloroquine was applied and cells further incubated for 2–6 h. When cells had grown to 80% confluence, transfection was done using the calcium phosphate method, with a final concentration of 0.125 M CaCl₂, 3 μg of pMD2. G, 5 μg of pRSV-Rev, 5 μg of pMDLg/pRRE and 10 μg of the respective pLL transfer vector. After overnight incubation, cells were further grown with fresh DMEM, 10% FCS and each 1% Penicillin and Streptomycin. Supernatant was collected on the following 2 days, filtered with a 0.45 μm syringe filter (Corning, Germany) and concentrated by ultracentrifugation for 2 h at 50.000 g (MLA-55, Beckman Coulter). Pellets were re-suspended in PBS and stored at −80 °C. Viral titer was determined using the QuickTiter^™ Lentivirus Titer Kit (Cell Biolabs, USA) according to the manufacturer’s instructions. Experimental vectors (HA-CREB, HA-CREB^S133A, shChrm1) were always prepared in parallel with the corresponding control vectors.