100 kda centrifugal filter device

OMVs were isolated and quantified as described45 (link) with minor modifications. P. aeruginosa strains were grown overnight in TSB and diluted 1:100 in fresh TSB broth containing 100 μM FeCl₃ until OD₆₀₀ reached 2.5 at 37 °C under agitation. After the bacteria were pelleted at 6,000g for 20 min, the supernatant fraction was filtered through a 0.45-μm vacuum filter, and the filtrate was concentrated with ultrafiltration (100 kDa centrifugal filter device (Millipore)). The retentate was filtered again through a 0.22-μm vacuum filter to remove any remaining bacteria. The resulting filtrate was subjected to ultracentrifugation at 265,000g for 60 min in a Beckman Coulter 70.1 rotor. For quantitative purposes, OMV preparations were subjected to two buffer (50 mM HEPES, pH 8.0) exchanges with 100-kDa centrifugal filter devices (Millipore). Protein content of the OMVs, which has been previously used to standardize OMV preparations, was used as a measure of OMV levels by evaluating the absorbance at 220 nm.

HucMSCs and medium were purchased from NUWACELL Co., Ltd. (Anhui, China). A 3D FloTrix miniSpin bioreactor (Beijing CytoNiche Biotechnology Co., Ltd., Beijing, China) was used for the 3D dynamic culture of hucMSCs, which were placed into the bioreactor at 37 °C in a humidified atmosphere with 5% CO₂.
The hucMSCs culture medium was collected before being centrifuged at 2000× g for 10 min and then at 10,000× g for 30 min. The supernatant was subjected to a 0.22 μm filter membrane (Millipore, Billerica, MA, USA), followed by a 100 kDa centrifugal filter device (Millipore, Billerica, MA, USA) and centrifuged at 4000× g for 20 min and then at 100,000× g for 70 min. All centrifugation steps were performed at 4 °C.
The hucMSC-EVs were identified by using Transmission Electron Microscopy (TEM) (Hitachi HT-7700, Tokyo, Japan), Nanoparticle Tracking Analysis (ZetaView PMX 110, Particle Metri, Meerbusch, Germany), and Western blotting analysis. Antibodies and dilution ratios are available in Supplementary Materials Table S1.