Quantifying Receptor-Mediated Phagocytosis and Inflammatory Responses to Fungi in Immune Cells

Patawee Asamaphan, Gordon D. Brown, Janet A. Willment*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

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 languageEnglish
Title of host publicationMethods in Molecular Biology
EditorsElaine Bignell
Place of PublicationNew York
PublisherHUMANA PRESS INC
Pages155-178
Number of pages24
Volume2260
ISBN (Electronic)978-1-0716-1182-1
ISBN (Print)978-1-0716-1181-4
DOIs
Publication statusPublished - 7 Jan 2021

Publication series

NameMethods in Molecular Biology
Volume2260
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

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

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