Related Experiment Video
Updated: Nov 22, 2025
Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry
Published on: December 7, 2019
Quantifying Receptor-Mediated Phagocytosis and Inflammatory Responses to Fungi in Immune Cells
Patawee Asamaphan1,2, Gordon D Brown1,3, Janet A Willment4,5
1Medical Research Council Centre for Medical Mycology at the University of Aberdeen, Aberdeen Fungal Group, Institute of Medical Sciences, Foresterhill, Aberdeen, UK.
Abstract:
Phagocytosis and cytokine production are important processes by which innate immune cells, especially professional phagocytes such as neutrophils and macrophages, control and regulate immunity to fungi. These cellular responses are initiated when conserved pathogen components, known as pathogen-associated molecular patterns (PAMPs), are recognized by pattern-recognition receptors (PRRs), which include members of the C-type lectin receptor (CLR) family that are able to bind to fungal cell wall-derived carbohydrates. Phagocytosis and cytokine production can be quantitatively examined by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively, using in vitro based assays with primary-derived murine cells and cell lines. Here, we describe a flow cytometry-based method using transduced cell lines to assess the ability of CLRs to mediate internalization, using A. fumigatus conidia and the β-1,3 glucan receptor, Dectin-1 (CLEC7A), as an example. The use of ELISA-based assays to measure cytokine production by immune cells that are induced in response to fungi and methods for isolating and culturing primary macrophages from various murine tissues are described.
Insights
This study presents methods to analyze fungal immunity. Researchers developed flow cytometry and ELISA assays to measure phagocytosis and cytokine production by immune cells, aiding fungal disease research.
Area of Science:
- Immunology
- Microbiology
Background:
- Innate immune cells like neutrophils and macrophages use phagocytosis and cytokine production to manage fungal infections.
- Pathogen-associated molecular patterns (PAMPs) on fungi are recognized by pattern-recognition receptors (PRRs), including C-type lectin receptors (CLRs), which bind fungal carbohydrates.
- Flow cytometry and ELISA are established methods for quantifying these cellular responses in vitro.
Purpose of the Study:
- To describe a flow cytometry-based method for assessing C-type lectin receptor (CLR) mediated internalization of fungal components.
- To detail enzyme-linked immunosorbent assay (ELISA) methods for measuring fungal-induced cytokine production.
- To outline procedures for isolating and culturing primary murine macrophages.
Main Methods:
- A flow cytometry assay using transduced cell lines to evaluate CLR-mediated internalization of Aspergillus fumigatus conidia.
- Utilizing Dectin-1 (CLEC7A) as a model β-1,3 glucan receptor.
- Employing ELISA to quantify cytokine production in immune cells stimulated by fungal components.
- Describing techniques for primary macrophage isolation and culture from murine tissues.
Main Results:
- The study successfully demonstrates a flow cytometry method to assess CLR-mediated phagocytosis of fungal elements.
- Established protocols for measuring fungal-induced cytokine release via ELISA.
- Provided detailed methods for obtaining and maintaining primary immune cells for experimental use.
Conclusions:
- The described methods provide robust tools for studying the innate immune response to fungi.
- These assays facilitate the investigation of C-type lectin receptor function in fungal recognition and clearance.
- The protocols support research into fungal immunity and the development of antifungal strategies.
