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

## TL;DR

This paper provides a detailed protocol to isolate and quantify large aging neutrophil-derived vesicles from both mouse and human samples using centrifugation and FACS.

## Contribution

The study introduces an optimized FACS-based protocol for isolating LAND-Vs from multiple biological sources in mice and humans.

## Key findings

- LAND-Vs can be effectively isolated from in vitro and ex vivo murine sources using centrifugation and FACS.
- The protocol is adaptable for human neutrophil-derived vesicles from isolated cells and blood.
- The method supports downstream functional and analytical studies of LAND-Vs.

## 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

•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

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.