Bacterial extracellular vesicle purification scheme. Bacterial cultures are grown and spun gently. (i) The supernatant is collected and filtered to remove cells. (ii) The supernatant is subsequently concentrated (~12X) using a Tangential Flow Filtration system with a 100 kDa-cutoff filter and the retentate is then filtered through a 0.22 µm pore. (iii) The concentrated filtrate is pelleted at high speed (iv) and the vesicle pellet resuspended in Optiprep solution. (v) The resuspended pellet is placed in the bottom of a 12.5 mL ultracentrifuge tube and overlaid with sequential decreasing percentages of Optiprep solutions. (vi) After ultracentrifugation, fractions are collected starting from the top of the gradient and analyzed for protein and lipid content.