Deep phenotyping of human neutrophils in whole blood using a 33-color spectral flow cytometry panel

Abstract Neutrophils are the most abundant leukocytes in the circulation and critical players in host defense and inflammation. They respond rapidly to numerous biological, chemical, and physical stimuli, making it challenging to characterize their steady-state phenotypes, activation states, and subsets in an unbiased and precise manner. To address this problem, we designed a 33-color spectral flow cytometry panel for the deep profiling of unprocessed neutrophils in human blood. This panel allows the profiling of neutrophil phenotypes related to activation, immune modulation, granule release, ontogeny, phagocytic capacity, and migration, in addition to monitoring all major human leukocyte populations. We validated the panel using whole blood stimulations that induce distinct phenotypic shifts in the neutrophil population. This optimized spectral flow cytometry panel allows comprehensive immune profiling of the functional heterogeneity of human blood neutrophils and is suitable for longitudinal or exploratory analysis of neutrophil dynamics and activation states in clinical cohorts.