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Published on: February 9, 2024
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Multiparametric Profiling of Neutrophil Function via a High-Throughput Flow Cytometry-Based Assay
Kyle D Timmer1, Daniel J Floyd1, Allison K Scherer1,2
1Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA 02114, USA.
Cells
|March 11, 2023
Summary
We developed a novel assay to simultaneously measure four key neutrophil functions: phagocytosis, reactive oxygen species generation, ectodomain shedding, and granule release. This tool aids in understanding neutrophil responses to pathogens and drug effects.
Area of Science:
- Immunology
- Cell Biology
Background:
- Neutrophils are crucial innate immune cells for pathogen clearance.
- Understanding neutrophil dysfunction in disease and drug side effects is critical.
- Existing assays often assess neutrophil functions individually.
Purpose of the Study:
- To develop a high-throughput, multi-functional flow cytometry assay for neutrophils.
- To simultaneously measure phagocytosis, ROS generation, ectodomain shedding, and granule release.
- To assess neutrophil responses to pathogens and immunomodulatory agents.
Main Methods:
- A single-reaction, microtiter plate-based flow cytometry assay was developed.
- Four fluorescent markers with minimal spectral overlap were utilized.
- Assay performance was validated using Candida albicans, inflammatory cytokines, and kinase inhibitors.
Main Results:
- The assay successfully detected changes in four canonical neutrophil functions.
- Inflammatory cytokines differentially modulated ectodomain shedding, phagocytosis, and degranulation.
- Kinase inhibitors (Btk, Syk, Src) suppressed all measured functions, which were restored by LPS co-stimulation.
Conclusions:
- This novel assay enables simultaneous assessment of multiple neutrophil effector functions.
- It allows for the identification of distinct neutrophil subpopulations and their activities.
- The assay can be used to study intended and off-target effects of immunomodulatory drugs on neutrophils.
