Combined phenotyping and functional analysis of isolated human natural killer cells using spectral flow cytometry

Natural Killer (NK) cells are potent effectors of the innate immune system, whose cytotoxicity relies on the balance of activating and inhibitory signals they receive. The diverse array of NK cell receptors that integrate these signals plays a key role in defining the many functional and developmental NK cell subsets. However, identifying these subsets is challenging due to receptor diversity, necessitating large and complex flow cytometric panels. Designing such panels can be further complicated by the substantial co-expression of markers, making fluorophore selection difficult due to spectral overlap and compensation constraints. Moreover, simple NK cell phenotyping does not directly correlate with their function or response to specific stimuli, limiting insights into NK cell behavior. To address these challenges, we present a degranulation assay protocol using tumor cells, followed by a 31-marker spectral flow cytometry panel on purified human NK cells. This approach enables the simultaneous screening of activating and inhibitory receptor expression, exhaustion markers, cytokine production and degranulation (via CD107a). By providing a comprehensive framework, this method supports a variety of research applications.