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An improved spectrofluorometric assay for quantitating yeast phagocytosis in cultures of murine peritoneal
1Department of Medical Microbiology and Immunology, College of Medicine, University of South Florida, Tampa 33612.
Abstract:
An accurate means by which to quantitate phagocytosis in murine resident peritoneal macrophages was developed by improving upon existing methods for measuring this important cellular function. Heat-killed Saccharomyces cerevisiae were conjugated to fluorescein isothiocyanate (FITC) and added to macrophage cultures. Following removal of uningested yeast, the macrophages were lysed and the fluorescence associated with the lysates was quantitated. The SEM were rarely +/- 10%. The improved assay was utilized to demonstrate the suppression of yeast phagocytosis by dexamethasone as measured by our radiometric assay. The spectrofluorometric assay produced results similar to those observed when the radiometric assay was employed to determine steroid induced suppression of yeast phagocytosis. However, the improved spectrofluorometric assay is more accurate, reliable, easier to perform, cost and time efficient, and a much safer method for quantitating yeast phagocytosis.
Insights
A new spectrofluorometric assay accurately quantifies phagocytosis in macrophages. This improved method for measuring yeast phagocytosis is more efficient and safer than previous techniques.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Phagocytosis is a critical cellular process for immune defense.
- Existing methods for quantifying phagocytosis in macrophages have limitations.
Purpose of the Study:
- To develop an improved, accurate, and efficient method for quantifying phagocytosis in murine resident peritoneal macrophages.
Main Methods:
- Developed a spectrofluorometric assay using fluorescein isothiocyanate (FITC)-conjugated heat-killed Saccharomyces cerevisiae.
- Quantitated fluorescence in lysed macrophage cultures after removing uningested yeast.
Main Results:
- The assay demonstrated suppression of yeast phagocytosis by dexamethasone.
- Results were comparable to a radiometric assay but with improved accuracy and efficiency.
Conclusions:
- The improved spectrofluorometric assay offers a more accurate, reliable, cost-effective, and safer method for quantifying yeast phagocytosis.
- This assay is suitable for studying factors affecting phagocytosis, such as steroid-induced suppression.