Cytofluorometric analysis of cell death in primary human neutrophils

Polymorphonuclear neutrophils (PMNs) are among the first effector cells recruited to the sites of infection, where they play a pivotal role in pathogen clearance and the resolution of inflammation. After phagocytosis and elimination of pathogens, PMNs typically undergo apoptosis and are subsequently removed by macrophages via phagocytosis. Due to their short lifespan, the timely clearance of PMNs is essential for maintaining immune homeostasis. However, stimuli, such as cytokines and bacterial products can prolong PMN survival, and this delayed apoptosis has been implicated in the pathogenesis of chronic inflammation. Elucidating the mechanisms that regulate PMN lifespan is therefore critical for the development of effective therapeutic strategies. Here we present a streamlined protocol for the isolation of primary human PMNs from venous blood and assessing their purity among intact singlet cells by flow cytometry using CD66b surface expression. We also describe methods for evaluating PMN viability with APC Annexin V/7-AAD or SYTOX™ Green staining. The protocol is easily adaptable to other viability dyes, such as propidium iodide, and can be extended to analyze additional PMN surface markers, including CD11b/Integrin alpha M, by flow cytometry.