Abstract
Bacterial extracellular vesicles (bEVs) participate in regulating both intra-bacterial community interactions and bacteria-host cell interactions. Here, we propose a universal and highly reproducible protocol for the extraction of bEVs from bacterial culture. The workflow combines differential centrifugation, ultracentrifugation, and enrichment, allowing efficient impurity removal and bEV enrichment. Nano-flow cytometry (nFCM) characterization of the extracted bEVs further confirmed the efficacy of this approach. For complete details on the use and execution of this protocol, please refer to Xie et al.1.