Purification protocol established for M. smithii EVs. Crude EVs were obtained by ultracentrifugation of cell-free supernatants from M. smithii cultures. The pelleted crude EVs were resuspended in 45% OptiPrep, and layers of lower concentrations were loaded on top. After ultracentrifugation of the OptiPrep gradient, fractions containing EVs were collected and combined. The collected EVs were washed in 10 times the volume of PBS solution and collected by ultracentrifugation. The pelleted purified EVs were then resuspended in PBS buffer. The schematic was partly created using graphical templates from Servier Medical Art under a Creative Commons license CC BY 4.0 ( https://creativecommons.org/licenses/by/4.0/ ). The illustration was created with Corel Draw Graphics Suite 2021.
