Wx floor ultra centrifuge

For EV preparation, BM-MSCs and iPSC-MSCs at 70–80% confluence in 17% FBS αMEM medium were transferred to the chemically defined and protein-free medium based on CD-CHO medium (Invitrogen, cat. number 10743-002) as reported [20 (link)]. After 6 hr, the medium was discarded and replaced by fresh medium and recovered at 48 hr to isolate EVs. Briefly, the conditioned medium was centrifuged at 2565g for 15 min to remove cells and debris, and then EVs were isolated from the supernatant by ultracentrifugation at 100,000g for 90 minutes at 4°C using Sorvall WX Floor Ultra Centrifuge with AH-629 36 ml swinging Bucket Rotor (Thermo). EV pellets were dissolved in cold TM buffer (pH 8.6) overnight at 4°C and frozen at −80°C in TM buffer (pH 8.6) containing 1% sucrose and 1% glycerol. The size and concentration of EVs were analyzed using the NanoSight LM 10 Nanoparticle Tracking Analysis System (Malvern). The NanoSight instrument was calibrated with polystyrene latex 100 nm and 200 nm microbeads (NTA4088 and NTA4089). Samples were measured under a range of a particle count from 2 × 10⁸ to 1 × 10⁹ particles per milliliter.

For EV isolation, the conditioned medium was filtered at 0.22 μm to remove cellular debris, and then EVs were isolated from the supernatant by ultracentrifugation at 100,000× g for 16 h at 4 °C using Sorvall WX Floor Ultra Centrifuge with AH-629 36 mL swinging Bucket Rotor (Thermo Fisher Scientific, Waltham, MA, USA). Isolated EVs were resuspended with PBS at concentrations of 5 to 10 × 10¹⁰/mL. The particle size and number of EVs were analyzed using the NanoSight LM 10 Nanoparticle Tracking Analysis System (Malvern, Malvern, UK). For in vivo biodistribution assays, iEVs were labeled with a near-infrared fluorescent dye, DiR (ThermoFisher), as reported in [17 (link),43 (link)]. To determine types of iEV recipient cells, iEVs were labeled with a fluorescent dye, PKH26 (Sigma), as reported in [44 (link)]. Splenocytes were isolated from NOD.B10.H2^b mice, cultured with RMPI 1640 culture medium (Gibco, Billings, MT, USA) containing 5% FBS, treated with 3 × 10⁹ particles/mL PKH26-labeled iEVs, and then examined with flow cytometry for PKH26 signal and markers of macrophages, T cells, or B cells as detailed below.