Related Experiment Video
Updated: Jan 3, 2026
09:22
High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization
Published on: November 27, 2014
15.2K
Using Imaging Flow Cytometry to Quantify Neutrophil Phagocytosis.
Asya Smirnov1, Michael D Solga2, Joanne Lannigan3
1Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, USA. as2kp@virginia.edu.
Methods in Molecular Biology (Clifton, N.J.)
|November 16, 2019
Summary
This study introduces a novel imaging flow cytometry method to precisely quantify how neutrophils bind and internalize bacteria like Neisseria gonorrhoeae, improving innate immunity research.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Neutrophils are key innate immune cells, acting as professional phagocytes crucial for pathogen defense and inflammation resolution.
- Quantifying neutrophil phagocytic capacity traditionally relies on microscopy, which can be labor-intensive and less high-throughput.
- Accurate measurement of bacterial binding and uptake by neutrophils is vital for understanding host-pathogen interactions and immune responses.
Purpose of the Study:
- To develop and validate an imaging flow cytometry (IFC) protocol for quantifying Neisseria gonorrhoeae binding and uptake by human neutrophils.
- To provide a high-throughput, quantitative method that overcomes limitations of traditional microscopy for neutrophil phagocytosis assays.
- To establish a versatile protocol adaptable for various particle types and multiplex analysis with neutrophil markers.
Main Methods:
- Developed an IFC protocol using fluorescently labeled Neisseria gonorrhoeae (Tag-it Violet™) and differential antibody staining (DyLight™ 650) for bound bacteria.
- Utilized primary adherent human neutrophils for the phagocytosis assay.
- Employed image analysis algorithms, including spot counting, to quantify bacterial association and internalization by neutrophils.
Main Results:
- The IFC method enables high-throughput, quantitative analysis of tens of thousands of neutrophils per condition.
- Quantified key parameters: percent of neutrophils associated with bacteria, percent of neutrophils with internalized bacteria, and the percentage of internalized bacteria.
- Demonstrated the protocol's ability to differentiate between bound and internalized bacteria accurately.
Conclusions:
- Imaging flow cytometry offers a powerful, quantitative, and high-throughput approach to study neutrophil phagocytosis.
- This protocol provides a robust method for assessing Neisseria gonorrhoeae uptake by neutrophils, applicable to various pathogens and particles.
- The adaptable nature of the protocol allows for multiplexing with neutrophil surface and intracellular markers, enhancing its utility in immunological research.
