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Protocol to isolate and quantify large aging neutrophil-derived vesicles.

Alan Y Hsu1, Qingxiang Huang2, Sizhou Feng2

  • 1Department of Pathology, PhD Program in Immunology, Harvard Medical School, Mass General Brigham, Dana-Farber/Harvard Cancer Center, Boston, MA 02115, USA.

STAR Protocols
|June 12, 2025
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Summary

This study details a protocol for isolating large aging neutrophil-derived vesicles (LAND-Vs) from mouse and human samples. The method uses centrifugation and fluorescence-activated cell sorting (FACS) for purification.

Keywords:
Cell BiologyCell isolationFlow CytometryImmunology

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Area of Science:

  • Cell Biology
  • Immunology
  • Extracellular Vesicles

Background:

  • Neutrophils are key immune cells involved in inflammation and host defense.
  • Aging neutrophils release large aging neutrophil-derived vesicles (LAND-Vs), which are significant in biological processes.
  • Understanding LAND-Vs requires effective isolation and purification methods.

Purpose of the Study:

  • To present a detailed protocol for the identification and purification of murine large aging neutrophil-derived vesicles (LAND-Vs).
  • To describe the isolation and quantification of human LAND-Vs.
  • To provide a reproducible method for researchers studying neutrophil extracellular vesicles.

Main Methods:

  • Isolation of murine LAND-Vs from in vitro neutrophil cultures, bone marrow, blood, and bronchoalveolar lavage fluid (BALF).
  • Purification of LAND-Vs using differential centrifugation and fluorescence-activated cell sorting (FACS).
  • Adaptation of the protocol for isolation and quantification of human LAND-Vs from neutrophils and blood.

Main Results:

  • Successful identification and purification of murine LAND-Vs using the described protocol.
  • Demonstration of LAND-Vs isolation from various ex vivo sources.
  • Established methodology for human LAND-Vs isolation and quantification.

Conclusions:

  • The presented protocol enables efficient isolation and purification of murine and human LAND-Vs.
  • This method facilitates further research into the biological roles of LAND-Vs.
  • The protocol is applicable to both in vitro and ex vivo samples, enhancing its utility.