Methods of Bacterial Membrane Vesicle Production, Purification, Quantification, and Examination of Their Immunogenic Functions

Bacterial membrane vesicles (BMVs) released by Gram-negative and Gram-positive bacteria are a bona fide secretion system that enable the dissemination of bacterial effector molecules, and can trigger a range of responses in the host. The study of BMV production, composition, and functions can give insights into their roles in mediating bacterial survival, pathogenesis, and disease. Furthermore, BMVs can be harnessed to develop cutting-edge nano-therapeutics including targeted chemotherapy delivery, antimicrobials, and novel vaccines. Here we describe routine methods that can be used for small- or large-scale production, isolation, and purification of outer membrane vesicles produced by Gram-negative bacteria, and membrane vesicles produced by Gram-positive bacteria, which we collectively refer to as BMVs. We discuss methods that can be used to visualize BMVs by electron microscopy, and to quantify their DNA, RNA, and protein cargo. We outline a method for the fluorescent labeling of BMVs that can be applied to examine their ability to interact with and enter host cells using a range of in vitro and in vivo biological assays. Finally, we provide a cell culture-based method that can be used to examine a range of immunogenic properties of BMVs, including their cytotoxicity, ability to activate pathogen-recognition receptors (PRRs), induce autophagy and cytokine responses, and modulate cellular pathways.