Protocol for autofluorescence-driven isolation of human peripheral blood eosinophils

Most common techniques for isolating eosinophils utilize CD16-negative selection, neglecting the CD16-positive fraction of eosinophils. Here, we present a protocol for isolating human CD16+ and CD16- eosinophils based on their autofluorescence using the MACSQuant Tyto cell sorter. We describe steps for purifying eosinophils and assessing purity, viability, and functional activity. We then detail procedures for Giemsa staining and the assessment of CD16 expression on human eosinophils. This protocol yields eosinophil purities of over 99%.