Dear Victor Guimarães

Best Overall Setup (if budget allows):

• Instrument: Beckman Optima XPN-100 (floor-standing)
• Rotors: SW 41 Ti (swinging-bucket) + 70 Ti (fixed-angle)
• Tubes: 13.2–70 mL polyallomer or polypropylene tubes depending on rotor and volume needs

This combination offers maximum flexibility, reproducibility, and compatibility with most published EV isolation protocols. If cost is a concern, Thermo Sorvall WX 80+ is a solid alternative.

Thank you for the answer!

Dear Dr. Victor Guimarães

Q1. Which model of ultracentrifuge (floor-standing or benchtop) is most appropriate for these purposes?

For isolating vesicles, a floor-standing ultracentrifuge is generally preferred over a benchtop model. This is because floor-standing centrifuges typically offer larger rotor capacities and higher speeds, which are often necessary for efficient vesicle isolation from large sample volumes, especially when using methods like differential ultracentrifugation. These floor-standing centrifuges generally have larger rotors, allowing for processing of multiple samples simultaneously or larger individual sample volumes. Moreover, they are designed to achieve higher speeds and generate greater centrifugal forces, crucial for isolating extracellular and intracellular vesicles from complex biological mixtures.

On the other hand, benchtop ultracentrifuges can also be used for isolating extracellular and intracellular vesicles, but they may have limitations in handling larger sample volumes or achieving the optimal speeds and forces required for certain methods.

Q2. What type of rotor (swinging-bucket or fixed-angle) is best suited for vesicle isolation?

For the isolation of extracellular and intracellular vesicles, fixed-angle rotors are generally preferred over swinging-bucket rotors because the tubes are fixed at a specific angle during centrifugation. As a result the pellet formed at the bottom of the tube is generally more condensed.

On the other hand, in swinging-bucket rotors, the tubes are held horizontally during centrifugation, and they swing to a vertical position during the run. in swinging-bucket rotors, the longer sedimentation path length can sometimes lead to lower pelleting efficiency compared to fixed-angle rotors.

So, for the isolation of extracellular and intracellular vesicles, the benefits of fixed-angle rotors in terms of pelleting efficiency and condensed pellet formation outweigh the advantages of swinging-bucket rotors.

Q3. What tube sizes and volumes are compatible and most commonly used in these protocols?

In these protocols, 50 mL conical tubes are mostly used. These tubes are suitable for collecting conditioned medium from cells and for performing low-speed centrifugation to get rid of large debris. For further isolation, you may use ultracentrifugation in which 14-38.5 mL ultracentrifuge tubes are made use of.

Q4. Are there specific brands or models (e.g., Beckman, Thermo/Sorvall, Hitachi) that are particularly well-suited for laboratories working with extracellular vesicles?

There is no such specific brand or model, but I would prefer Beckman while working with extracellular vesicles (EVs). It is frequently cited in research protocols for EV isolation. Beckman Coulter provides a range of ultracentrifuges, including the Optima L-100XP and Optima TLX, specifically designed for EV isolation. It offers high performance, reliability, and compatibility with various rotor options, making it a strong candidate for research purpose in this field. This is just my suggestion.

Regards,

Malcolm Nobre