Protocol for isolating extracellular vesicles from alveolar macrophages phagocytosing MRSA in vitro cell culture models

Extracellular vesicles (EVs) play a crucial role in delivering bioactive cargo in infectious diseases. Here, we present a protocol for isolating EVs from alveolar macrophages (AMs) that phagocytose methicillin-resistant Staphylococcus aureus (MRSA) in vitro cell culture models. We describe steps for bacterial preparation; infection of AMs with MRSA; and isolation, purification, and characterization of EVs. This protocol provides a valuable perspective for studying EVs derived from pathogen-infected immune cells. For complete details on the use and execution of this protocol, please refer to Bai et al.1 [Display omitted] •Optimization of the process of alveolar macrophage infection by MRSA•Isolation of EVs from alveolar macrophage that phagocytose MRSA in vitro•Purification of EVs by iodixanol density gradient fractionation•Characterization of EVs by TEM, NTA, and western blotting Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Extracellular vesicles (EVs) play a crucial role in delivering bioactive cargo in infectious diseases. Here, we present a protocol for isolating EVs from alveolar macrophages (AMs) that phagocytose methicillin-resistant Staphylococcus aureus (MRSA) in vitro cell culture models. We describe steps for bacterial preparation; infection of AMs with MRSA; and isolation, purification, and characterization of EVs. This protocol provides a valuable perspective for studying EVs derived from pathogen-infected immune cells.