Beckman tube

For EV isolation using UC, a Beckman Optima L-100 XP ultracentrifuge with a SW 28 rotor (Beckman Coulter, Brea, CA, USA) was used. The FF supernatant was ultracentrifuged in Beckman tubes (No. 326823; Beckman Coulter) at 100,000× g for 70 min at 4 °C to pellet EVs. Following the first ultracentrifugation, the supernatant was discarded, and freshly filtered phosphate-buffered saline (PBS; No. 18912-014; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) was added to wash the pellet. The suspension was ultracentrifuged one more time at 100,000× g for 70 min at 4 °C to pellet the extracellular vesicles. The final pellet was resuspended in 100 µL of PBS and kept at −80 °C until further analysis.

After 48 h, we collected the culture medium from the bottom wells in order to determine the amount of exosomes that could have passed through the membrane filter during the incubation period. The media collected from the bottom wells (6 ml of media total, from 3 identical wells) with or without addition of exosomes were collected and subjected to exosome isolation [30 (link)]. Briefly, the media were transferred to 15 ml Beckman tubes (Beckman Coulter, Indianapolis, IN; catalog number #342082) and centrifuged at 2000 g for 30 min to sediment contaminating cells. Clean cell-free media (6 ml each) were transferred into fresh 15 ml centrifuge tubes and 3 ml of exosome isolation reagent was added (Invitrogen, Carlsbad, CA; catalog number 4478359) and mixed by inverting the tubes three times. Exosomes were precipitated by incubating the mix overnight at 4 °C and collected by centrifuge at 10,000 g for 1 h at 4 °C. Supernatant was removed by aspiration and the exosome pellet was suspended in 100 μl of PBS and stored at −80 °C until use.

For polysome fractionation, B cells were incubated with cyclohexamide (CHX, Sigma Aldrich, 100 μg ml⁻¹) for 3 min at 37 °C prior collection. After washing the cells with ice-cold PBS containing CHX (100 μg ml⁻¹), cell pellets were either stored at −80 °C or resuspended in polysome buffer (300 mM NaCl, 15 mM MgCl₂, 15 mM Tris-HCl; pH 7.5, 100 μg ml⁻¹ cycloheximide CHX, 100 U RnaseOUT from Life Technologies) containing 1% Triton X-100. After incubation on ice for 10–15 s, cells were spun at 17,000 × g at 4 °C. Cytoplasmic cell supernatant was placed at the top of a 50–10% sucrose gradient prepared previously in 17 ml- Beckman tubes (by underlying, from the bottom to the top, 3 ml of 10, 20, 30, 40 and 50% sucrose solutions made up in polysome buffer). Then, samples were centrifuged at 250,000 × g for 2hr at 4 ˚C (acceleration 7, deceleration 7) using a pre-cooled SW41Ti. Samples were kept at 4 °C at all times. One millilitre fractions were automatically collected using gradient fractionator after injecting a 65% sucrose solution (made up in polysome buffer containing Bromophenol blue as a tracer dye) to the bottom of the tube. Polysome fractions were mixed with 3 ml of 7.7 M guanidine HCl and 4 ml of 100% ethanol, and incubated overnight at −20 °C.