Protocol for the isolation of human neutrophil-derived extracellular vesicles for characterization and functional experimentation
Small extracellular vesicles (EVs) derived from neutrophils play an important role in the remodeling of extracellular matrices in human health and disease. Here, we present a protocol for isolating EVs secreted from human neutrophils either ex vivo or within circulation. We describe steps for neutrophil and plasma isolation, neutrophil activation, and EV isolation and enumeration. We then detail procedures for isolating CD66b<sup>+</sup> EVs using magnetic bead pull-down and subsequent characterization by flow cytometry, western blot, and neutrophil elastase activity assays. For complete information on the generation and use of this protocol, please refer to Genschmer et al.<sup>1</sup>.
Authors
- Delores A. Stacks (ORCID: https://orcid.org/0000-0002-3824-0953)
- Kristopher R. Genschmer (ORCID: https://orcid.org/0000-0002-1836-9522)
- Dakota C. Finley
- Robert P. Richter (ORCID: https://orcid.org/0000-0002-3753-1994)
- Ningyong Xu (ORCID: https://orcid.org/0000-0003-3483-3097)
- Julian A. Smith (ORCID: https://orcid.org/0000-0003-1244-4277)
- Camilla Margaroli (ORCID: https://orcid.org/0000-0003-3952-0778)
- Ezgi Sari (ORCID: https://orcid.org/0000-0003-4818-6126)
- Crystal Lewis
- Tamiajoi Langster
Institutions
- University of Alabama at Birmingham (US)
Publication Details
- Journal
- STAR Protocols
- Published
- 2025-04-06
- DOI
- https://doi.org/10.1016/j.xpro.2025.103739
- Citations
- 2
- Primary Topic
- Extracellular vesicles in disease
- Type
- article
- Field-Weighted Citation Impact
- 1.61
Funders
- National Institutes of Health
- National Heart, Lung, and Blood Institute
- National Institute of General Medical Sciences