70ti rotor

U1 cART EVs were isolated via ultracentrifugation. First, supernatants were centrifuged at 1200 rpm for five minutes to pellet any cellular debris. The resulting supernatant was then centrifuged at 100,000×g for 90 min using the 70Ti rotor (Beckman). The pellet was washed with PBS followed by another centrifugation at 100,000×g for 90 min. EV pellets were resuspended in PBS and then frozen at − 20 °C for downstream analysis.
A549 cells were infected with 5.0 × 10⁵ TCID50 of human coronavirus OC43 for 96 h. Supernatants were collected and centrifuged at 2000 rpm for five minutes, filtered through a 0.22 µm sterile filter, and then centrifuged at 100,000×g for 90 min using the 70Ti rotor (Beckman). The pellet was resuspended in sterile PBS and treated with 45 mJ/cm³ of UV(C) LED irradiation. Samples of UV(C)-treated EVs (1, 5, 10 µL) were added to Vero cells for two passages and no viral outgrowth was detected (data not shown). Remaining EVs were frozen at − 20 °C for downstream analysis.
U1 cART and OC43 EVs were analyzed via NTA using the ZetaView Z-NTA (Particle Metrix). Prior to analysis the equipment was calibrated per the manufacturer’s recommendation and each EV sample was diluted in deionized water prior to analysis. The acquired data was analyzed by the instrument’s built-in software.

E. coli was grown in LB broth (500 mL, 37 °C, 180 rpm) until reaching an OD600 nm value of 1. Bacterial cells were removed through centrifugation at 3000× g for 20 min at 4 °C. Residual bacteria and cellular debris were eliminated by sequential 0.45 and 0.22 µm filtration (VWR Scientific, Bridgeport, NJ, USA). OMVs were collected by centrifugation at 100,000× g for 2 h at 4 °C (Optima XPN-100 Beckman Coulter and rotor 70Ti, Palo Alto, CA, USA). The vesicles were washed in sterile PBS1X through ultracentrifugation and re-suspended in 150 μL of PBS1X. LB agar plates were used to check the sterility of the OMVs. The purified vesicles were stored at −20 °C until use [28 (link)].

OMV purification was performed following the protocol used by Martora et al. with some slight modifications [13 (link)]. Briefly, three K. pneumoniae strains were grown in LB broth (600 mL, 37 °C, 180 rpm) to an OD₆₀₀ nm value of 1. The bacterial cells were decanted through centrifugation and supernatants were filtered at 0.45 µm and 0.22 µm (Millex-GS filters, Millipore, Darmstadt, Germany). The cell-free supernatants were centrifuged at 100,000× g for 2 h at 4 °C (centrifuge Optima XPN-100 Beckman Coulter and rotor 70Ti, Palo Alto, CA, USA). The pellets were washed in sterile PBS1X by ultracentrifugation and re-suspended in 200 µL of PBS1X. The sterility of the OMVs was checked on LB agar plates. The purified OMVs were stored at −20°C after dynamic light scattering (DLS) analysis.