Abstract
Neutrophils play a crucial role in maintaining lung health by defending against infections and participating in inflammation processes. Here we describe a detailed protocol for evaluating pulmonary neutrophil phenotype using a murine model of sterile inflammation induced by the fungal cell wall particle zymosan. We provide step-by-step instructions for the isolation of single cells from both lung tissues and airspaces, followed by comprehensive staining techniques for both cell surface markers and intracellular components. This protocol facilitates the sorting and detailed characterization of lung neutrophils via flow cytometry, making it suitable for downstream applications such as mRNA extraction, single-cell sequencing, and analysis of neutrophil heterogeneity. We also identify and discuss essential considerations for conducting successful neutrophil flow cytometry experiments. This work is aimed at researchers exploring the intricate functions of neutrophils in the lung under physiological and pathological conditions with the aid of flow cytometry.