1. Quantifying Receptor-Mediated Phagocytosis and Inflammatory Responses to Fungi in Immune Cells.
- Author
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Asamaphan P, Brown GD, and Willment JA
- Subjects
- Animals, Aspergillus fumigatus immunology, Aspergillus fumigatus pathogenicity, Host-Pathogen Interactions, Lectins, C-Type genetics, Macrophages immunology, Macrophages metabolism, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, RAW 264.7 Cells, Transduction, Genetic, Aspergillus fumigatus metabolism, Cytokines metabolism, Enzyme-Linked Immunosorbent Assay, Flow Cytometry, Inflammation Mediators metabolism, Lectins, C-Type metabolism, Macrophages microbiology, Phagocytosis
- 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.
- Published
- 2021
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