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Protocol to isolate and quantify large aging neutrophil-derived vesicles

  • 1Department of Pathology, PhD Program in Immunology, Harvard Medical School, Mass General Brigham, Dana-Farber/Harvard Cancer Center, Boston, MA 02115, USA. Electronic address: ahsu7@bwh.harvard.edu.
  • 2State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.
  • 3Department of Pathology, PhD Program in Immunology, Harvard Medical School, Mass General Brigham, Dana-Farber/Harvard Cancer Center, Boston, MA 02115, USA. Electronic address: hrluo@bwh.harvard.edu.
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June 12, 2025

Abstract

Large aging neutrophil-derived vesicles (LAND-Vs) are ∼1 μm ectosomes released by aging neutrophils. Here, we present a protocol to identify and purify murine LAND-Vs from in vitro neutrophil cultures and ex vivo sources, including bone marrow, blood, and bronchoalveolar lavage fluid (BALF), using centrifugation and fluorescence-activated cell sorting (FACS). We also describe steps for isolation and quantification of human LAND-Vs from isolated neutrophils and human blood. For complete details on the use and execution of this protocol, please refer to Hsu et al.1.

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