Protocol to isolate and quantify large aging neutrophil-derived vesicles

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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 • Optimized FACS protocol to isolate LAND-Vs from neutrophils, blood, and BALF • Isolated LAND-Vs are suitable for comprehensive downstream analyses and functional assays • Detailed sample preparation and gating strategy for LAND-V identification Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.

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