Purification and Characterization of Extracellular Vesicles

In some experiments, C-DGUC was performed as described previously with some modifications [34 (link),55 (link)]. In brief, gut-lavage fluids were pooled from sham or CLP mice (10 mice in each group) and concentrated into a final volume of 30 mL using the Amicon ultra-15 centrifugal filter unit (Merck) per the manufacturer’s instructions. The concentrated samples were placed on 0.8 M and 2 M sucrose cushions and centrifuged at 100,000× g for 120 min at 4 °C in as SW32Ti rotor using the L-80 ultracentrifuge (Beckman Coulter). The interface layer was harvested, diluted five-fold with HEPES buffered saline (HBS), filtered through the 0.22-μm filter unit (Merck) and then repeated for sucrose ultracentrifugation in a SW41Ti rotor as described above. The interface layer was harvested and diluted in 2.3 mL of HBS and mixed with an equal volume of 60% iodixanol to achieve a 30% iodixanol concentration containing EVs. This solution was overlaid with 20% and 5% iodixanol solutions and centrifuged at 200,000× g for 120 min at 4 °C in an SW32Ti rotor. Ten consecutive 1-mL fractions were diluted in PBS and centrifuged at 100,000× g for 120 min to pellet the EVs. Each pellet was resuspended in 100 µL of PBS and the protein concentration was measured as described above.