Max xp ultracentrifuge

After preconditioning, cells were washed with PBS three times and cultured in serum-free DMEM without phenol red supplemented with 1% penicillin/streptomycin and 1% insulin–transferrin–selenium (ITS, Thermo Fisher Scientific) for 48 h. After this time, the conditioned medium was collected, centrifuged at 1000×g for 10 min at 4 °C, 5000×g for 20 min at 4 °C to remove dead cells and debris, and, subsequently filtered through 0.45- and 0.22-µm filters. After these steps, the secretome fraction was concentrated by centrifugation at 4000×g for 40 min at 4 °C, using a 3 kDa MWCO Amicon® Ultra device (Merck-Millipore, MA, USA). For EV isolation, the concentrated secretome was ultracentrifuged at 110,000×g for 2 h in a MAX-XP ultracentrifuge equipped with a TLA-45 fixed-angle rotor (Beckman Coulter, Krefeld, Germany), and the resulting pellet was washed with 0.1 µm filtered PBS (1 ml) and centrifuged again with the same settings. Finally, the supernatant was removed, and isolated EVs were suspended in 50–100 µl of filtered PBS and stored at − 80 °C for further analysis. When samples from different donors were pooled, concentrated secretome samples (AMICON samples) were equally mixed based on protein concentration (n = 5 donors) and EVs were isolated by ultracentrifugation as described.

V. fischeri cultures were grown at 28 °C in LBS until late exponential phase (OD ≃ 3). The cells were removed by low-speed centrifugation (8,000g) at 4 °C, and the culture supernatant was filtered through a 0.22-μm-pore-size PVDF membrane filter (Millipore). OMVs were then collected from the filtered supernatant by centrifugation for 2 h in a TLA-45 rotor using a Max-XP ultracentrifuge (Beckman Coulter) at 180,000g and 4 °C. The pelleted OMVs were washed by resuspension in Dulbecco’s phosphate-buffered saline (DPBS) with added salt (0.4 M NaCl) [38 (link)] and re-centrifugation at 200,000g for 1 h at 4 °C using either a MLA-50 or TLA-110 rotor in an Optima-XP centrifuge (Beckman Coulter). The resulting pellets were resuspended in saline DPBS and filter-sterilized through 0.45-μm-pore-size PVDF membrane filter (Millipore) before storing at −80 °C. Before OMVs were added to live squid, they were further purified with a sucrose density gradient as previously described [38 (link)]. To estimate the OMV concentration, total protein of the sample was determined with the Qubit Protein Assay Kit (Invitrogen).