An improved spectrofluorometric assay for quantitating yeast phagocytosis in cultures of murine peritoneal macrophages.
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.
Ragsdale
R L
RL
Department of Medical Microbiology and Immunology, College of Medicine, University of South Florida, Tampa 33612.
Grasso
R J
RJ
D000818:Animals; D003907:Dexamethasone / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D008264:Macrophages / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010587:Phagocytosis / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000276:immunology; D013050:Spectrometry, Fluorescence / Q000379:methods; D013861:Thiocyanates
D005452:Fluoresceins; D013861:Thiocyanates; D003907:Dexamethasone; D016650:Fluorescein-5-isothiocyanate