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.
Original language | English |
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Title of host publication | Methods in Molecular Biology |
Editors | Elaine Bignell |
Place of Publication | New York |
Publisher | HUMANA PRESS INC |
Pages | 155-178 |
Number of pages | 24 |
Volume | 2260 |
ISBN (Electronic) | 978-1-0716-1182-1 |
ISBN (Print) | 978-1-0716-1181-4 |
DOIs | |
Publication status | Published - 7 Jan 2021 |
Publication series
Name | Methods in Molecular Biology |
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Volume | 2260 |
ISSN (Print) | 1064-3745 |
ISSN (Electronic) | 1940-6029 |
Bibliographical note
Funding Information:This work was supported by the Wellcome Trust (102705, 97377) and the MRC Centre for Medical Mycology and the University of Aberdeen (MR/N006364/1).
Publisher Copyright:
© 2021, Springer Science+Business Media, LLC, part of Springer Nature.
Keywords
- A. fumigatus
- Alveolar macrophages
- Bone marrow-derived macrophages
- C-type lectin receptors
- Dectin-1
- Fungi
- Inflammatory responses
- Peritoneal macrophages
- Phagocytosis
- RAW264.7 cells
- TNF