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Quantitative analysis of phagocytosis of Cryptococcus neoformans by adherent phagocytic cells by fluorescence
A M Walenkamp1, J Scharringa, F M Schramel
1The Eijkman-Winkler Institute for Microbiology, Infectious Diseases and Inflammation, Division of Vaccines, University Hospital Utrecht, The Netherlands.
Abstract:
Macrophages and monocytes are adherent phagocytic cells which play an important role in host defence against the yeast-like fungus Cryptococcus neoformans. Before, phagocytosis by adherent phagocytes could only be measured by means of microscopy or by a radioactive assay, which both have obvious disadvantages. We have developed a new, rapid and objective method to measure phagocytosis of C. neoformans by adherent phagocytes (e.g. alveolar macrophages) using a fluorescence multi-well plate reader. This method allows us to discriminate accurately between adherence and internalisation of C. neoformans by macrophages during long term incubation. In addition, the method was used to study the role of the mannose receptor in phagocytosis of the acapsular yeast in the absence of serum by human monocyte-derived macrophages (MDM).
Insights
A new fluorescence assay accurately measures Cryptococcus neoformans phagocytosis by macrophages. This method improves upon older techniques and helps study the mannose receptor
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Macrophages and monocytes are key phagocytic cells in host defense against Cryptococcus neoformans.
- Traditional methods for measuring phagocytosis (microscopy, radioactive assays) have significant limitations.
- A need exists for a more efficient and objective method to quantify phagocytosis.
Purpose of the Study:
- To develop and validate a novel, rapid, and objective fluorescence-based assay for measuring C. neoformans phagocytosis by adherent phagocytes.
- To differentiate between adherence and internalization of C. neoformans by macrophages over time.
- To investigate the role of the mannose receptor in C. neoformans phagocytosis by human monocyte-derived macrophages (MDM) in serum-free conditions.
Main Methods:
- Development of a fluorescence-based assay using a multi-well plate reader.
- Quantification of C. neoformans phagocytosis by adherent phagocytes, including alveolar macrophages.
- Application of the assay to study human monocyte-derived macrophages (MDM) and the mannose receptor.
Main Results:
- The new fluorescence assay provides a rapid and objective measurement of C. neoformans phagocytosis.
- The method accurately distinguishes between fungal adherence and internalization by macrophages.
- The assay was successfully employed to study the mannose receptor's role in phagocytosis by MDM.
Conclusions:
- A novel fluorescence assay offers a superior method for quantifying C. neoformans phagocytosis by macrophages.
- This assay facilitates detailed studies on macrophage-pathogen interactions and receptor-mediated phagocytosis.
- The findings contribute to understanding host defense mechanisms against fungal infections.