Open top thinwall ultra clear tube

We previously developed a method for isolating EVs from plasma samples (Karimi et al., 2018 (link)), and this was applied in the current study for comparing the composition of EVs in serum and plasma. Briefly, a 50% iodixanol (OptiPrep™, Sigma Aldrich) working solution was prepared and used to further prepare 30% and 10% iodixanol solutions. Next, 6‐ml plasma or serum was layered on top of 2‐ml 50%, 2‐ml 30% and 2‐ml 10% iodixanol solutions (13.2 mL, Open‐Top Thinwall Ultra‐Clear Tube, product no: 344059, Beckman Coulter) before being ultracentrifuged at 178,000 × g_avg for 2 h at 4°C (SW 41 Ti rotor, k‐factor 143.9, Beckman Coulter). A visible EV‐enriched band with a volume of 1 ml was collected from the 30% and 10% interface and then loaded onto a home‐made SEC column packed with Sepharose CL‐2B (GE Healthcare, Uppsala, Sweden) in a Telos SPE column (Kinesis, Cambridgeshire, UK) as previously described in Ax et al. (2020 (link)) and Karimi et al. (2018 (link)). The collection of 0.5‐ml fractions immediately begun when the sample was added. In total, 30 fractions of 0.5 ml were eluted and collected using 0.2‐μm‐filtered PBS as the elution buffer.

E. cava (12 g) was dissolved in 360 mL of DW with continuous stirring for 24 h at 50 °C. The mixture was then centrifuged at 3000× g for 30 min at room temperature to remove large debris or particulate matter. The resulting supernatant (approximately 38.5 mL) was transferred into an Open-Top Thinwall Ultra-Clear Tube (Beckman Coulter, Brea, CA, USA) and purified by centrifugation at 50,000× g for 90 min.
The supernatant, enriched with smaller particles including exosomes, was collected and subjected to ultracentrifugation at 100,000× g for 120 min at room temperature to precipitate the exosomes. The resulting exosome pellet was washed by resuspending in DW and again ultracentrifuged at 100,000× g for 120 min at room temperature.
The obtained pellet comprised E. cava-derived exosomes (EV-EC). The final exosome suspension was resuspended in DW and subjected to freeze-drying.

Plasma with or without DNase dedicated for sEVs isolation was further processed by differential UC (Figure 6). Briefly, the first UC was at 100,000× g for 90 min in a Type 70.1 Ti Fixed-Angle Titanium Rotor (Beckman Coulter Optima L-90K Ultracentrifuge, Beckman Coulter, Inc., Brea, CA, USA). After UC, the supernatant was discarded, and the pellet was resuspended in 90 μL phosphate buffer saline (PBS) (3 × 30 μL). The second UC was with discontinuous (12–36%) iodixanol-density gradient that was prepared using a commercial OptitPrep solution (Sigma-Aldrich, St. Louis, MO, USA). The collected pellets were placed at the bottom of an Open-Top Thinwall Ultra-Clear Tube (Beckman Coulter, Inc., Brea, CA, USA), and a solution of a descending concentration of the iodixanol density gradient was layered on top [20 (link)]. The samples were ultracentrifuged at 120,000× g for 15 h in an SW 32.1 Ti Swinging-Bucket Rotor (Beckman Coulter Optima L-90K Ultracentrifuge, Beckman Coulter, Inc., Brea, CA, USA) [20 (link)]. After UC, the upper fractions of the gradient were collected at a volume of 8 mL, pipetted into fusible ultracentrifuge tubes and supplemented with PBS solution. The third UC was at 120,000× g for 4 h in a Type 70.1 Ti Fixed-Angle Titanium Rotor. The resulting pellet was collected by pipetting in 90 μL PBS (3 × 30 μL PBS). All UC steps were performed at 4 °C.