Protocol for autofluorescence-driven isolation of human peripheral blood eosinophils
Year: 2024
Type: article
Abstract: 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%. • Instructions for purifying eosinophils based on autofluorescence using cell sorting • Steps to assess eosinophil purity and viability by flow cytometry and Giemsa staining • Guidance on performing an eosinophil activation test to confirm functional activity • Manual for analyzing CD16 expression on purified eosinophils Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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%.
Source: STAR Protocols
Institutions: Carl von Ossietzky Universität Oldenburg, Klinikum Oldenburg
Language: English
Subfield: Physiology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $1,500
Funder: Deutsche Forschungsgemeinschaft
Awards: G1421102612, G4308171117, Zellsortierer
OpenAlex ID: W4404496369