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Monitoring internalization of Histoplasma capsulatum by mammalian cell lines using a fluorometric microplate assay
1Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison 53706, USA.
Abstract:
We have developed a fluorometric microtiter plate assay to quantify the internalization of Histoplasma capsulatum yeasts by macrophages. The assay utilizes the fluorescent dye Calcofluor White to label the yeast cell wall and the vital dye trypan blue, which does not enter viable macrophages, to quench fluorescence of extracellular labeled yeasts. Murine RAW 264.7 cells showed more efficient internalization of strain G217B yeasts than human U937 cells. Both cell lines exhibited a dependence upon actin, and, to a lesser degree, microtubules, in G217B uptake.
Insights
A new assay quantifies Histoplasma capsulatum yeast uptake by macrophages. Murine cells showed higher yeast internalization than human cells, dependent on actin and microtubules.
Area of Science:
- Mycology
- Cell Biology
- Immunology
Background:
- Histoplasma capsulatum is an opportunistic fungal pathogen.
- Macrophage phagocytosis is crucial for controlling fungal infections.
Purpose of the Study:
- To develop and validate a novel fluorometric assay for quantifying yeast-macrophage internalization.
- To compare yeast uptake efficiency between murine and human macrophage cell lines.
Main Methods:
- A fluorometric microtiter plate assay was developed.
- Calcofluor White dye labeled yeast cell walls.
- Trypan blue quenched extracellular yeast fluorescence.
Main Results:
- The assay successfully quantified Histoplasma capsulatum yeast internalization by macrophages.
- Murine RAW 264.7 cells demonstrated more efficient yeast uptake than human U937 cells.
- Yeast internalization was dependent on actin and, to a lesser extent, microtubules in both cell lines.
Conclusions:
- The developed assay provides a reliable method for studying yeast-macrophage interactions.
- Differential uptake efficiency suggests distinct macrophage responses to fungal pathogens.
- Cytoskeletal elements play a significant role in fungal yeast internalization.